BACKGROUND: The PI3K/Akt/mTOR pathway and autophagy are important physiological processes. But their roles in eCRSwNP remains controversial.
METHODS: In this study, we used the eCRSwNP mouse model, PI3K/Akt/mTOR pathway inhibitors, and autophagy inhibitors and activators to investigate the regulatory effects of the PI3K/Akt/mTOR pathway on autophagy, and their effects on eosinophilic inflammation, and tissue remodeling. The role of ILC2s in eCRSwNP was also studied, and the relationship between ILC2s and autophagy was preliminarily determined.
RESULTS: Our results show that eosinophilic inflammation in eCRSwNP mice could be inhibited by promoting the autophagy; otherwise, eosinophilic inflammation could be promoted. Meanwhile, inhibition of the PI3K/Akt/mTOR pathway can further promote autophagy and inhibit eosinophilic inflammation. Meanwhile, inhibiting the PI3K/Akt/mTOR pathway and promoting autophagy can reduce the number of ILC2s and the severity of tissue remodeling in the nasal polyps of eCRSwNP mice.
CONCLUSIONS: We conclude that the PI3K/Akt/mTOR pathway plays roles in eosinophilic inflammation and tissue remodeling of eCRSwNP, in part by regulating the level of autophagy. The downregulation of autophagy is a pathogenesis of eCRSwNP; therefore, the recovery of normal autophagy levels might be a new target for eCRSwNP therapy. Furthermore, autophagy might inhibit eosinophilic inflammation and tissue remodeling, in part by reducing the number of ILC2s.

Cell-based cancer immunotherapy has achieved significant advancements, providing a source of hope for cancer patients. Notwithstanding the considerable progress in cell-based immunotherapy, the persistently low response rates and the exorbitant costs associated with their implementation still present a formidable challenge in clinical settings. In the landscape of cell-based cancer immunotherapies, an uncharted territory involves Type 2 innate lymphoid cells (ILC2s) and interleukin-33 (IL-33) which promotes ILC2 functionality, recognized for their inherent ability to enhance immune responses. Recent discoveries regarding their role in actuating cytolytic T lymphocyte responses, including curbing tumor growth rates and hindering metastasis, have added a new dimension to our understanding of the IL-33/ILC2 axis. These recent insights may hold significant promise for ILC2 cell-based immunotherapy. Nevertheless, the prospect of adoptively transferring ILC2s to confer immune protection against tumors has yet to be investigated. The present study addresses this hypothesis, revealing that ILC2s isolated from the lungs of tumor-bearing mice, and tumor infiltrating ILC2s when adoptively transferred after tumor establishment at a ratio of one ILC2 per sixty tumor cells, leads to an influx of tumor infiltrating CD4+ and CD8+ T lymphocytes as well as tumor infiltrating eosinophils resulting in a remarkable reduction in tumor growth. Moreover, we find that post-adoptive transfer of ILC2s, the number of tumor infiltrating ILC2s is inversely proportional to tumor size. Finally, we find corollaries of the IL-33/ILC2 axis enhancing the infiltration of eosinophils in human prostate carcinomas patients\' expressing high levels of IL-33 versus those expressing low levels of IL-33. Our results underscore the heightened efficacy of adoptively transferred ILC2s compared to alternative approaches, revealing an approximately one hundred fifty-fold superiority on a cell-per-cell basis over CAR T-cells in the specific targeting and elimination of tumors within the same experimental model. Overall, this study demonstrates the functional significance of ILC2s in cancer immunosurveillance and provides the proof of concept of the potential utility of ILC2 cell-based cancer immunotherapies.

BACKGROUND: Recent 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.
METHODS: A 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.
RESULTS: The 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.
CONCLUSIONS: This 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.

Herein we described a retrospective analysis of a 13-year-old female patient with facial dysmorphia and immune disorder caused by BCL11B gene mutation. The patient upon physical examination presented a particular face (thin eyebrows, small mandible, and widened eye distance), delayed language and motor development. Supplementary examination showed expansion of CD8+, absence of type 2 Innate Lymphoid Cells, increased IgG and altered distribution of T cells. Genetic testing revealed a heterozygous frameshift variation in exon 4 of the BCL11B gene; c.1887_c.1893delCGGCGGG (p.Gly630Glyfs*91). Finally, a BCL11B gene mutation could lead to abnormal development of the nervous and immune systems, therefore, it is necessary to consider this syndrome in patients with the clinical and immunological phenotype described below.

Down-regulation of chloride transporter SLC26A3 or down-regulated in adenoma (DRA) in colonocytes has recently been linked to the pathogenesis of ulcerative colitis (UC). Because exaggerated immune responses are one of the hallmarks of UC, these current studies were undertaken to define the mechanisms by which loss of DRA relays signals to immune cells to increase susceptibility to inflammation.
NanoString Immunology Panel, fluorescence assisted cell sorting, immunoblotting, immunofluorescence, and quantitative real-time polymerase chain reaction assays were used in wild-type and DRA knockout (KO) mice. Interleukin (IL)-33 blocking was used to determine specific changes in immune cells and co-housing/broad spectrum antibiotics administration, and ex vivo studies in colonoids were conducted to rule out the involvement of microbiota. Colonoid-derived monolayers from healthy and UC patient biopsies were analyzed for translatability.
There was a marked induction of Th2 (>2-fold), CD4+ Th2 cells (∼8-fold), RORγt+ Th17, and FOXP3+ regulatory T cells (Tregs). DRA KO colons also exhibited a robust induction of IL-33 (>8-fold). In vivo studies using blocking of IL-33 established that T2 immune dysregulation (alterations in ILC2, Th2, and GATA3+ iTregs) in response to loss of DRA was due to altered epithelial-immune cell crosstalk via IL-33.
Loss of DRA in colonocytes triggers the release of IL-33 to drive a type 2 immune response. These observations emphasize the critical importance of DRA in mucosal immune homeostasis and its implications in the pathogenesis of UC.

T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted \"Th2 cells\" or \"ILC2s\" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient \"Th2\" cells and \"ILC2s\" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient \"ILC2s\" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.

A characteristic feature of sarcoidosis is a dysregulated immune response to persistent stimuli, often leading to the formation of non-necrotizing granulomas in various organs. Although genetic susceptibility is an essential factor in disease development, the etiology of sarcoidosis is not fully understood. Specifically, whether autoimmunity contributes to the initiation or progression of the disease is uncertain. In this study, we investigated systemic autoimmunity to vimentin in sarcoidosis. IgG antibodies to human vimentin were measured in sera from sarcoidosis patients and healthy controls. Mice immunized with recombinant murine vimentin were challenged intravenously with vimentin-coated beads to mimic pulmonary sarcoidosis. Lungs from treated mice were studied for cellular infiltration, granuloma formation, and gene expression. Immune cells in the bronchoalveolar lavage fluid were evaluated by flow cytometry. Compared to healthy controls, sarcoidosis patients had a higher frequency and levels of circulating anti-vimentin IgG. Vimentin-immunized mice developed lung granulomas following intravenous challenge with vimentin-coated beads. These sarcoidosis-like granulomas showed the presence of Langhans and foreign body multinucleated giant cells, CD4 T cells, and a heterogeneous collection of MHC II positive and arginase 1-expressing macrophages. The lungs showed upregulated pro-inflammatory gene expression, including Ifng, Il17, and Tnfa, reflecting TH1/TH17 responses typical of sarcoidosis. In addition, genes in the TH2 canonical pathway were also upregulated, congruent with increased numbers of ILC2 in the bronchoalveolar lavage. Overall, these results further validate vimentin as an autoantigen in sarcoidosis and provide evidence for an anti-vimentin immune response in disease pathogenesis. Our study also highlights the possible role of ILC2-driven TH2-like responses in the formation of lung granulomas in sarcoidosis.

Type 2 innate lymphoid cells (ILC2s) exert an increasingly important influence on the pathological process of allergic rhinitis (AR), which is affected by microRNAs-mediated post-transcriptional regulation. This study aims to investigate the function of miR-150-5p in AR patients and the mouse model of AR. The mouse model of AR was established using the OVA challenge. The expressions of miR-150-5p, ICAM-1, p-p38 and p-GATA-3 were evaluated via RT-qPCR and western blot analysis. The level of ILC2s was examined with flow cytometry. Concentrations of OVA-specific IgE, IL-13 and IL-5 in serum were evaluated using ELISA. Histopathological examination was conducted through H&E staining. The interplay between ICAM-1 and miR-150-5p was determined through the DLR assay. The decreased miR-150-5p expression and increased ICAM-1, p-p38 and p-GATA-3 expressions and ILC2s levels were detected in AR patients and AR mice compared with controls. Treatment with miR-150-5p lentivirus alleviated AR symptoms (sneezing, rubbing, mucosa inflammation, serum type 2 cytokines and OVA-specific IgE) and lowered the ILC2s level in AR mice. MiR-150-5p was found to directly bind to 3\'-UTR of ICAM-1 and downregulate ICAM-1 expression, thereby descending the level of p-p38, p-GATA-3 and suppressing ILC2s function to alleviate AR symptoms. Treatment with Lenti-ICAM-1 counteracted these protective effects of miR-150-5p. Upregulation of miR-150-5p repressed the ICAM-1/p38 axis which was vital to ILC2s development and function, thereby alleviating allergic symptoms of AR.

Acute graft-versus-host disease (aGVHD) is one of the most common causes of morbidity for patients undergoing allogeneic stem cell transplantation. There is preliminary evidence that activated Group 2 innate lymphoid cells (ILC2s) from wild type (WT) mice reduces the lethality of aGVHD and is effective in treating lower gastrointestinal (GI) tract manifestations of aGVHD. This raises the prospect that ILC2s may be used for cell-based therapy of aGVHD but vigorous investigation is necessary to assess their impacts on different aspects of aGVHD. Genetically engineered mice which either express Id1 protein (Id1tg/tg), an inhibitor of E protein transcription factors or have E protein genes knocked out (dKO) in the thymus produce massive numbers of ILC2s, thus allowing extensive evaluation of ILC2s. We investigated whether these ILC2s have protective effects in aGVHD as WT ILC2s do using an established mouse model of aGVHD.
bone marrow transplant was performed by irradiating BALB/c strain of recipient mice and transplanting with bone marrow and T cells from the MHC-disparate C57BL/6 strain. We isolated ILC2s from Id1tg/tg and dKO mice and co-transplanted them to study their effects. Our results confirm that activated ILC2s have a protective role in aGVHD, but the effects varied depending on the origin of ILC2s. Co-transplantation of ILC2s from Id1tg/tg mice were beneficial in aGVHD and are especially helpful in ameliorating the skin manifestations of aGVHD. However, ILC2s from dKO mice were less effective at the protection and behaved differently depending on if the cells were isolated from dKO mice were pre-treated with IL-25 in vivo.
These findings support the notion that thymus-derived ILC2s from Id1tg/tg mice are protective against aGVHD, with a significant improvement of skin lesions and they behave differently from dKO mice in the setting of aGVHD.

UNASSIGNED: Type 2 innate lymphoid cells (ILC2s) have emerged as key players in the development of type 2 driven diseases such as allergy and asthma. Due to their low number in the circulation, in vitro expansion is needed to unravel their mechanisms of action.
UNASSIGNED: The aim of this study is to assess the impact of different culture conditions and address whether the method of expansion may distinctly affect healthy donor or patient-derived ILC2s.
UNASSIGNED: Here, we described the impact of six different culture conditions on the proliferation, phenotype and function of human ILC2s freshly obtained from healthy donors (healthy ILC2s) and allergic patients (patient ILC2s).
UNASSIGNED: We showed that the cytokine cocktail or the PHA induced the highest proliferation of healthy ILC2s and patient ILC2s, respectively. We observed that the stromal cells OP9, used as ILC2 feeders, did not boost their proliferation, but impaired the activation marker expression and the function of patient ILC2s. Furthermore, we demonstrated that the culture conditions differently impacted the activation state of c-Kithigh and c-Kitlow ILC2s, in both healthy donors and allergic patients. Last, we also observed that ILC2s expanded only with IL-2 and IL-7 were the most prone to secrete IL-5 and IL-13 upon IL-33 stimulation. In contrast, in patients, the addition of OP9 cells during the expansion restrained their type 2 cytokine secretory functions.
UNASSIGNED: This report highlights that culture conditions distinctly impacted on the healthy or patient ILC2 behavior, with important consequences for their study in disease settings.