Parallel origins and functions of T cells and ILCs.

Objective: To investigate the change of innate lymphoid cells (ILC) subsets in peripheral blood and ascites in liver cirrhotic patients complicated with spontaneous bacterial peritonitis (SBP). Methods: The data of 62 patients with liver cirrhosis admited to the Zhumadian Central Hospital from November 2019 to November 2020 were analyzed. Among them, 41 cases were complicated with untainted ascites (untainted ascites group), while the other 21 cases were complicated with SBP (SBP group). Meanwhile, 20 cases of controls who received healthy examination in the same period were also enrolled (control group). Peripheral blood mononuclear cell (PBMC) was isolated from peripheral blood of all patients and controls. Mononuclear cell in ascites was isolated from patients with liver cirrhosis. The percentage of ILC1, ILC2, and ILC3 subsets in PBMC and mononuclear cell in ascites were measured by flow cytometry. CD3-CD19-CD20-CD14- cells (lin-cells) were purified from ascites and were stimulated with lipopolysaccharide (LPS) for 24 h. The transcription factor T-bet, GATA3, and RORγt mRNA relative level in lin-cells was semi-quantified by real-time PCR. Cytokine level in the supernatants was measured by enzyme linked immunosorbent assay. Differences of ILC subsets in peripheral blood and ascites were compared among groups. Results: There were twenty-nine males and twelve females in untainted ascites group, aged M(Q1,Q3) 49(33, 78) years. There were twelve males and nine females in SBP group, aged 50(37, 76) years. There were eleven males and nine females in control group, aged 48(32, 69) years. lin-CD45+CD161+CD127+ ILC cells could be detected in both peripheral blood and ascites. There was no significant difference in total ILC percentage within PBMC among untainted ascites group, SBP group, and control group (P=0.235). There was also no significant difference of total ILC percentage within mononuclear cells in ascites between untainted ascites group and SBP group (P=0.232). The differences were not statistically significant of peripheral CD117-CRTh2-ILC1, CRTh2+ILC2, or CD117+CRTh2-ILC3 within peripheral ILC among untainted ascites group, SBP group, and control group (all P>0.05). ILC1 percentage in ascites was up-regulated in SBP group compared with untainted ascites group (35.69%±3.39% vs 26.40%±3.85%, P<0.001), while ILC2 in ascites was down-regulated in SBP group (36.83%±7.70% vs 48.35%±9.45%, P<0.001). There was no statistical difference in ILC3 percentage in ascites between the two groups (P=0.230). T-bet mRNA relative level and IFN-γ production by lin- cells from ascites were elevated in response to LPS stimulation in SBP group compared with untainted ascites group (both P<0.001). GATA3 mRNA relative level and IL-5/IL-13 secretion by lin-cells from ascites were reduced in SBP group compared with untainted ascites group (both P<0.05). There was no significant difference of RORγt mRNA relative level or IL-17/IL-22 expression between the two groups (both P>0.05). Conclusion: Peripheral ILC subsets do not change in liver cirrhosis patients with SBP. ILC1 percentage is up-regulated, and ILC2 percentage is down-regulated in ascites in liver cirrhosis patients with SBP.

RelB is a member of the NF-κB family, which is essential for dendritic cell (DC) function and maturation. However, the contribution of RelB to the development of allergic airway inflammation (AAI) is unknown. Here, we identify a pivotal role for RelB in the development of spontaneous AAI that is independent of exogenous allergen exposure. We assessed AAI in two strains of RelB-deficient (RelB-/-) mice: one with a targeted deletion and one expressing a major histocompatibility complex transgene. To determine the importance of RelB in DCs, RelB-sufficient DCs (RelB+/+ or RelB-/-) were adoptively transferred into RelB-/- mice. Both strains had increased pulmonary inflammation compared with their respective wild-type (RelB+/+) and heterozygous (RelB+/-) controls. RelB-/- mice also had increased inflammatory cell influx into the airways, levels of chemokines (CCL2/3/4/5/11/17 and CXCL9/10/13) and T-helper cell type 2-associated cytokines (IL-4/5) in lung tissues, serum IgE, and airway remodeling (mucus-secreting cell numbers, collagen deposition, and epithelial thickening). Transfer of RelB+/- CD11c+ DCs into RelB-/- mice decreased pulmonary inflammation, with reductions in lung chemokines, T-helper cell type 2-associated cytokines (IL-4/5/13/25/33 and thymic stromal lymphopoietin), serum IgE, type 2 innate lymphoid cells, myeloid DCs, γδ T cells, lung Vβ13+ T cells, mucus-secreting cells, airway collagen deposition, and epithelial thickening. These data indicate that RelB deficiency may be a key pathway underlying AAI, and that DC-encoded RelB is sufficient to restore control of this inflammation.

Cancer treatment has been revolutionized with the development of immunomodulatory therapies. These therapies have primarily focused on enhancing T-cell responses: whether it is unleashing T cells through blockade of regulatory checkpoint inhibitors or generation of chimeric antigen receptor (CAR) T cells. However, there has been recent interest in harnessing the immunotherapeutic potential of other cytotoxic cells such as Natural Killer (NK) cells. Similar to NK cells, innate lymphoid cells (ILCs) may offer another target of these immunotherapy approaches. Before the potential of these cells can be realized, there is a need for better understanding of these recently described cell populations. ILCs act as the immune system's first responders and have been shown to play a key role in tissue homeostasis, chronic inflammation and cancer. Three main groups of non-cytotoxic ILCs (ILC1, ILC2 and ILC3) have been broadly defined based on developmental trajectories and function, driven by expression of specific transcription factors. Deeper characterization of these cells through either high parameter protein analysis or single-cell RNA sequencing has revealed a more complex and heterogeneous nature of ILCs across different tissues and donors. Therefore, the identity of ILCs is still elusive and controversial. In this study, we developed a comprehensive approach to further refine the signatures of human circulating ILC subsets. Total ILCs (Lineage- CD127+ cells) were enriched from 4 normal donors by flow sorting using the BD FACSAria™ Fusion cell sorter and processed for downstream single-cell multiomic characterization. BD® AbSeq reagents and a targeted BD Rhapsody™ Immune Response Panel were used to enable simultaneous detection of 42 proteins and 399 genes using the BD Rhapsody™ Single-Cell Analysis System. Differential protein and gene expression analysis in addition to combinatorial expression of CD294 and CD117 confirmed 3 conventional ILC populations as well as the signatures of three distinct subsets within ILC1. This discovery approach provided information about relative expression of a small selection of proteins or surface marker-coding genes that enable the discrimination of these ILC subsets. These data were used to design high-parameter flow cytometry panels for high-throughput analysis of different healthy donors. ILC subsets differentially distributed across donors were detected and defined using unsupervised computational analysis confirming the result of multiomic analysis, while the functional and biological relevance of the identified subsets remains to be assessed. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 laser product. BD, the BD Logo, FACSymphony and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Aaron J. Tyznik, Mirko Corselli, Gisle V. Baracho, Chip Lomas, Silin Sa, Stephanie Widmann, Suraj Saksena, Smita Ghanekar. Resolving the heterogeneity of human circulating innate lymphoid cells via simultaneous, high-dimensional analysis of protein and gene expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3310.

Innate lymphoid cells (ILCs), comprising ILC1, 2, and 3 subpopulations, play unique roles in maintaining microbiome homeostasis, mucosal tissue integrity, and control of inflammation. So far, their characterization is dominantly based on tissue‐resident ILCs, whereas little information is available on circulating ILCs, in particular in newborns. In order to get a deeper understanding of neonatal innate immunity, we analyzed the transcriptomes and effector functions of cord blood (CB) ILCs. By RNAseq analysis, all ILC subsets could be clearly distinguished from each other. CB‐derived ILCs were generally closer related to neonatal T than natural killer (NK) cells and several factors shared by all three ILC subsets such as CD28, CCR4, and SLAMF1 are commonly expressed by T cells but lacking in NK cells. Notably, CB ILCs exhibited a unique signature of DNA binding inhibitor (ID) transcription factors (TF) with high ID3 and low ID2 expression distinct from PB‐ or tonsil‐derived ILCs. In vitro stimulation of sorted CB ILCs revealed distinct differences to tissue‐resident ILCs in that ILC1‐like and ILC3‐like cells were nonresponsive to specific cytokine stimulation, indicating functional immaturity. However, CB ILC3‐like cells expressed toll‐like receptors TLR1 and TLR2 and upon stimulation with the TLR2:1 ligand Pam3CSK4, responded with significantly increased proliferation and cytokine secretion. Together, our data provide novel insights into neonatal ILC biology with a unique TF signature of CB ILCs possibly indicating a common developmental pathway and furthermore a role of CB ILC3‐like cells in innate host defense.

Simple SummaryAnti-CTLA-4 and anti-PD-1 immune checkpoints inhibitors (ICI) have revolutionized the treatment of metastatic melanoma patients, leading to durable responses. However, some patients still not respond to this clinically used immunotherapies and there is a lack of biomarkers leading to the choice of first-line therapies. Innate lymphoid cells (ILC) express immune checkpoint receptors and are involved in anti-melanoma immune response. The aim of this article is to study ILCs from peripheral blood of melanoma patients receiving Ipilimumab, an anti-CTLA-4 treatment, and their association with clinical responses to this therapy. Our results show an impact of Ipilimumab on ILCs proportions and phenotype in blood. Moreover, the presence of anergic CD56dimCD16−DNAM-1− NK cells were associated with progression of the disease. These findings demonstrate the important role of ILC in the response to ICI.Immunotherapy targeting immune checkpoint receptors brought a breakthrough in the treatment of metastatic melanoma patients. However, a number of patients still resist these immunotherapies. Present on CD8+T cells, immune checkpoint receptors are expressed by innate lymphoid cells (ILCs), which may contribute to the clinical response. ILCs are composed of natural killer (NK) cells, which are cytotoxic effectors involved in tumor immunosurveillance. NK cell activation is regulated by a balance between activating receptors that detect stress molecules on tumor cells and HLA-I-specific inhibitory receptors. Helper ILCs (h-ILCs) are newly characterized ILCs that secrete cytokines and regulate the immune homeostasis of tissue. We investigated the modulation of blood ILCs in melanoma patients treated with ipilimumab. Circulating ILCs from metastatic stage IV melanoma patients and healthy donors were studied for their complete phenotypic status. Patients were studied before and at 3, 6, and 12 weeks of ipilimumab treatment. A comparison of blood ILC populations from donors and melanoma patients before treatment showed changes in proportions of ILC subsets, and a significant inverse correlation of CD56dim NK cells and h-ILC subsets was identified in patients. During treatment with ipilimumab, percentages of all ILC subsets were reduced. Ipilimumab also impacted the expression of the CD96/TIGIT/DNAM-1 pathway in all ILCs and increased CD161 and CTLA-4 expression by h-ILCs. When considering the response to the treatment, patients without disease control were characterized by higher percentages of CD56bright NK cells and ILC1. Patients with disease control displayed larger populations of activated CD56dimCD16+ DNAM-1+ NK cells, while anergic CD56dimCD16−DNAM-1− NK cells were prominent in patients without disease control. These results provide original findings on the distribution of ILC subsets in advanced melanoma patients and their modulation through immunotherapy. The effects of ipilimumab on these ILC subsets may critically influence therapeutic outcomes. These data indicate the importance of considering these innate cell subsets in immunotherapeutic strategies for melanoma patients.

Innate lymphoid cells (ILCs) are abundant in mucosal tissues and involved in tissue homeostasis and barrier function. Although several ILC subsets have been identified, it is unknown if additional heterogeneity exists, and their differentiation pathways remain largely unclear. We applied mass cytometry to analyze ILCs in the human fetal intestine and distinguished 34 distinct clusters through a t-SNE-based analysis. A lineage (Lin)-CD7+CD127-CD45RO+CD56+ population clustered between the CD127+ ILC and natural killer (NK) cell subsets, and expressed diverse levels of Eomes, T-bet, GATA3, and RORγt. By visualizing the dynamics of the t-SNE computation, we identified smooth phenotypic transitions from cells within the Lin-CD7+CD127-CD45RO+CD56+ cluster to both the NK cells and CD127+ ILCs, revealing potential differentiation trajectories. In functional differentiation assays, the Lin-CD7+CD127-CD45RO+CD56+CD8a- cells could develop into CD45RA+ NK cells and CD127+RORγt+ ILC3-like cells. Thus, we identified a previously unknown intermediate innate subset that can differentiate into ILC3 and NK cells.

Although substantial parallels have been made between transcription factor regulation of cytokine production by innate lymphoid cell (ILC) and Th cell subsets, we are still learning how ILC subsets are regulated during immune responses. Critical factors that promote ILC development and stimulate their effector functions have been identified, but mechanisms that control their homeostasis and downregulate their cytokine secretion remain poorly understood. In this review, we consider some of the potential positive and negative regulators of ILC homeostasis and function in physiological and pathological conditions.

Innate immune crosstalk in asthmatic airways: Innate lymphoid cells coordinate polarization of lung macrophages

Several distinct innate lymphoid cell (ILC) populations have been recently identified and shown to play a critical role in the immediate immune defense. In the context of tumors, there is evidence to support a dual role for ILCs with pro- or antitumor effects, depending on the ILC subset and the type of cancer. This ambivalent role has been particularly well-described in colorectal cancer models (CRC), but the presence and the evolution of ILCs in the peripheral blood of metastatic CRC (mCRC) patients have not yet been explored. Here, we investigated the distribution of ILC subsets in 96 mCRC patients who were prospectively included in the “Epitopes-CRC02” trial. Peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry at metastatic diagnosis and after 3-months of treatment. The treatments consisted of Oxaliplatin-based chemotherapies for 76% of the patients or Folfiri (5FU, Irinotecan) chemotherapies for 14% of patients. Compared to healthy donors, the frequency of total ILCs was dramatically increased at metastatic diagnosis. CD56+ ILC1-like cells were expanded, whereas ILC2, NCR− ILCP and NCR+ ILCP subsets were decreased. Combined analysis with the systemic anti-telomerase hTERT Th1 CD4 response revealed that patients with low anti-TERT Th1 CD4 responses had the highest frequencies of total ILCs at diagnosis. Of those, 91% had synchronous metastases, and their median progression-free survival was 7.43 months (vs. 9.17 months for the other patients). In these patients, ILC1 and ILC2 were significantly decreased, whereas CD56+ ILC1-like cells were significantly increased compared to patients with low frequency of total ILCs and high anti-TERT responses. After treatment, the NCR+ ILCP were further decreased irrespective of the chemotherapy regimen, whereas the balance between ILC1 and CD56+ ILC1-like cells was modulated mainly by the Folfiri regimen in favor of ILC1. Altogether our results describe the effects of different chemotherapies on ILCs in mCRC patients. We also establish for the first time a link between frequency of ILCs and anti-tumor CD4 T cell responses in cancer patients. Thus, our study supports an interest in monitoring ILCs during cancer therapy to possibly identify predictive biomarkers in mCRC.

Innate lymphoid cells (ILCs) develop from ILC progenitors in the bone marrow. Various ILC precursors (ILCPs) with different ILC subset lineage potentials have been identified based on the expression of cell surface markers and ILC-associated key transcription factor reporter genes. This study characterized an interleukin (IL)-7Rα+IL-18Rα+ ILC progenitor population in the mouse bone marrow with multi-ILC lineage potential on the clonal level. Single-cell gene expression analysis revealed the heterogeneity of this population and identified several subpopulations with specific ILC subset-biased gene expression profiles. The role of IL-18 signaling in the regulation of IL-18Rα+ ILC progenitors and ILC development was further investigated using Il18- and Il18r1-deficient mice, in vitro differentiation assay, and adoptive transfer model. IL-18/IL-18R-mediated signal was found to not be required for early stages of ILC development. While Il18r1-/- lymphoid progenitors were able to generate all ILC subsets in vitro and in vivo like the wild-type counterpart, increased IL-18 level, as often occurred during infection or under stress, suppressed the growth of ILCP/ILC in an IL-18Ra-dependent manner via inhibiting proliferation and inducing apoptosis.

Innate lymphoid cells (ILCs) have emerged as largely tissue-resident archetypal cells of the immune system. We tested the hypotheses that renal ischemia-reperfusion injury (IRI) is a contributing factor to polarization of ILCs and that glucocorticoid-induced leucine zipper (GILZ) and cannabidiol regulate them in this condition. Mice subjected to unilateral renal IRI were treated with the following agents before restoration of renal blood flow: cannabidiol, DMSO, transactivator of transcription- (TAT-) GILZ, or the TAT peptide. Thereafter, kidney cells were prepared for flow cytometry analyses. Sham kidneys treated with either cannabidiol or TAT-GILZ displayed similar frequencies of each subset of ILCs compared to DMSO or TAT, respectively. Renal IRI increased ILC1s and ILC3s but reduced ILC2s compared to the sham group. Cannabidiol or TAT-GILZ treatment of IRI kidneys reversed this pattern as evidenced by reduced ILC1s and ILC3s but increased ILC2s compared to their DMSO- or TAT-treated counterparts. While TAT-GILZ treatment did not significantly affect cells positive for cannabinoid receptors subtype 2 (CB2+), cannabidiol treatment increased frequency of both CB2+ and GILZ-positive (GILZ+) cells of IRI kidneys. Subsequent studies showed that IRI reduced GILZ+ subsets of ILCs, an effect less marked for ILC2s. Treatment with cannabidiol increased frequencies of each subset of GILZ+ ILCs, but the effect was more marked for ILC2s. Indeed, cannabidiol treatment increased CB2+ GILZ+ ILC2s. Collectively, the results indicate that both cannabidiol and GILZ regulate ILC frequency and phenotype, in acute kidney injury, and that the effects of cannabidiol likely relate to modulation of endogenous GILZ.

Innate lymphoid cells (ILCs) are a family of predominantly tissue-resident lymphocytes that critically orchestrate immunity, inflammation, tolerance and repair at barrier surfaces of the mammalian body. Heterogeneity among ILC subsets is comparable to that of adaptive CD4+ T helper cell counterparts, and emerging studies demonstrate that ILC biology is also dictated by cellular metabolism that adapts bioenergetic requirements during activation, proliferation or cytokine production. Accumulating evidence in mouse models and human samples indicates that ILCs exhibit profound roles in shaping states of metabolic health and disease. Here we summarize and discuss our current knowledge of the cell-intrinsic and cell-extrinsic metabolic factors controlling ILC responses, as well as highlight contributions of ILCs to organismal metabolism. It is expected that continued research in this area will advance our understanding of how to manipulate ILCs or their metabolism for therapeutic strategies that benefit human health.

Innate lymphoid cells (ILC) are a heterogeneous group of cellular subsets that produce large amounts of T cell-associated cytokines in response to innate stimulation in the absence of Ag. In this study, we define distinct patterns of surface marker and cytokine expression among the ILC subsets that may further delineate their migration and function. Most notably, we found that the subset previously defined as group 1 ILC (ILC1) contains CD4(+) CD8(-), CD4(-) CD8(+), and CD4(-) CD8(-) populations. Although all ILC1 subsets shared characteristics with Th1 cells, CD4(+) ILC1 also demonstrated significant phenotypic and functional heterogeneity. We also show that the frequencies of CD4(+) ILC1 and NKp44(+) group 3 ILC, but not CD4(-) ILC1 or group 2 ILC, are increased in the peripheral blood of individuals with systemic sclerosis (SSc), a disease characterized by fibrotic and vascular pathology, as well as immune dysregulation. Furthermore, we demonstrate that CD4(+) and CD4(-) ILC1 are functionally divergent based on their IL-6Rα expression and that the frequency of IL-6Rα expression on ILC is altered in SSc. The distinct phenotypic and functional features of CD4(+) and CD4(-) ILC1 suggest that they may have differing roles in the pathogenesis of immune-mediated diseases, such as SSc.

Innate lymphoid cells (ILCs) are a heterogenous population of the innate immune system, enriched at mucosal surfaces and are pivotal regulators of immune homeostasis. ILCs are the innate counterpart of T cells. Like T cells, ILC subsets are highly plastic with their composition and function controlled by alterations in their microenvironment. This plasticity allows for the trans-differentiation between the subsets to rapidly respond to their immune environment. The tumor microenvironment (TME) is a heterogeneous milieu characterized by different cytokines and growth factors. Through interaction with the tumor microenvironment, ILCs can transdifferentiate into different subsets resulting in pro or anti-tumor immunity. Thus, studying ILC plasticity might result in new therapeutic approaches for cancer therapy. In this review, we summarize current findings of the functional and plastic heterogeneity of ILCs in homeostasis as well as disease settings with a specific focus on cancer. We specifically highlight tumor-driven plasticity and how ILC-induced inflammation can impact the tumor microenvironment and anti-tumor immunity.

Innate lymphoid cells (ILCs) are emerging as critical modulators of inflammation in rheumatoid arthritis, contributing to both disease pathology and resolution. Group 3 ILCs (ILC3s) mirror TH17 cells in their production of IL-17A and IL-22, promoting fibroblast activation, neutrophil recruitment and synovial inflammatory cascades. By contrast, group 2 ILCs (ILC2s) engage reparative and immunoregulatory pathways via secretion of IL-9, IL-13 and IL-10. Lymphoid tissue inducer (LTi) ILCs contribute to ectopic lymphoid tissue neogenesis and stromal remodelling in early disease. Clinically, alterations in ILC subset composition correlate with disease activity, therapeutic responsiveness and inflammatory burden. Advances in high-dimensional immunophenotyping, spatial transcriptomics and single-cell multi-omics now enable precise mapping of ILC subsets and their effector programmes across peripheral blood and synovial tissue, supporting their use in biomarker discovery and treatment pipelines. Furthermore, modulation of ILCs by targeting upstream cytokines, signalling pathways or the use of microbiota-derived metabolites is a potential therapeutic strategy. Finally, cell-based avenues include IL-10-producing ILC2s (ILC210) and engineered chimeric antigen receptor (CAR)-ILC2s for targeted, tissue-resident immune modulation. Although still in the preclinical stages, these approaches highlight the translational potential of ILCs as biomarkers and therapeutic targets in rheumatoid arthritis.