Human CD127 negative ILC2s show immunological memory.

ILC2s are key players in type 2 immunity and contribute to maintaining homeostasis. ILC2s are also implicated in the development of type 2 inflammation-mediated chronic disorders like asthma. While memory ILC2s have been identified in mouse, it is unknown whether human ILC2s can acquire immunological memory. Here, we demonstrate the persistence of CD45RO, a marker previously linked to inflammatory ILC2s, in resting ILC2s that have undergone prior activation. A high proportion of these cells concurrently reduce the expression of the canonical ILC marker CD127 in a tissue-specific manner. Upon isolation and in vitro stimulation of CD127-CD45RO+ ILC2s, we observed an augmented ability to proliferate and produce cytokines. CD127-CD45RO+ ILC2s are found in both healthy and inflamed tissues and display a gene signature of cell activation. Similarly, mouse memory ILC2s show reduced expression of CD127. Our findings suggest that human ILC2s can acquire innate immune memory and warrant a revision of the current strategies to identify human ILC2s.

Aberrant characteristics of peripheral blood innate lymphoid cells in COPD, independent of smoking history.

Background: Distinguishing asthma and COPD can pose challenges in clinical practice. Increased group 1 innate lymphoid cells (ILC1s) have been found in the lungs and peripheral blood of COPD patients, while asthma is associated with elevated levels of ILC2s. However, it is unclear whether the inflammatory characteristics of ILC1s and ILC2s differ between COPD and asthma. This study aims to compare peripheral blood ILC subsets and their expression of inflammatory markers in COPD patients, asthma patients and controls. Methods: The study utilised multi-colour flow cytometry to analyse peripheral blood ILC populations in clinically stable COPD patients (n=38), asthma patients (n=37), and smoking (n=19) and non-smoking (n=16) controls. Results: Proportions of peripheral blood inflammatory CD4+ ILC1s were significantly higher in COPD patients than in asthma. Proportions of CD4- ILC1s were increased in COPD patients compared to asthma patients and smoking controls. Frequencies of CD117- ILC2s were significantly reduced in COPD patients compared with asthma patients. In contrast, the fraction of inflammatory CD45RO+ cells within the CD117- ILC2 population was significantly increased. Principal component analyses showed that combined features of the circulating ILC compartment separated COPD patients from asthma patients and both control groups. Conclusion: Our in-depth characterisation of ILC1 and ILC2 populations in peripheral blood revealed significant differences in their phenotypes between COPD and asthma patients and smoking or non-smoking controls. These findings suggest a role for both ILC subsets in COPD disease pathology, independent of smoking history, and may have implications for patient stratification and therapy development.

Type-2 CD8+ T-cell formation relies on interleukin-33 and is linked to asthma exacerbations.

CD4+ T helper 2 (Th2) cells and group 2 innate lymphoid cells are considered the main producers of type-2 cytokines that fuel chronic airway inflammation in allergic asthma. However, CD8+ cytotoxic T (Tc) cells - critical for anti-viral defense - can also produce type-2 cytokines (referred to as 'Tc2' cells). The role of Tc cells in asthma and virus-induced disease exacerbations remains poorly understood, including which micro-environmental signals and cell types promote Tc2 cell formation. Here we show increased circulating Tc2 cell abundance in severe asthma patients, reaching peak levels during exacerbations and likely emerging from canonical IFNγ+ Tc cells through plasticity. Tc2 cell abundance is associated with increased disease burden, higher exacerbations rates and steroid insensitivity. Mouse models of asthma recapitulate the human disease by showing extensive type-2 skewing of lung Tc cells, which is controlled by conventional type-1 dendritic cells and IFNγ. Importantly, we demonstrate that the alarmin interleukin-33 (IL-33) critically promotes type-2 cytokine production by lung Tc cells in experimental allergic airway inflammation. Our data identify Tc cells as major producers of type-2 cytokines in severe asthma and during exacerbations that are remarkably sensitive to alterations in their inflammatory tissue micro-environment, with IL-33 emerging as an important regulator of Tc2 formation.

Innate lymphoid cells: from helper to killer.

Five subsets of ILCs are extensively described, Lymphoid Tissue inducer (LTi) cells, cytotoxic NK cells and non-cytotoxic helper ILC1s, ILC2s and ILC3s. So far, the main focus has been on the potent cytokine production by helper ILCs and their plastic nature that allows them to switch function and phenotype upon environmental changes. Recent advances in the field indicate the presence of cytotoxic helper ILCs that are distinct from conventional NK cells. In humans, these cytotoxic ILCs can develop from conventional helper ILCs whereas in mice this remains to be elucidated. In this review we discuss the identification, development and function of cytotoxic helper ILCs subsets in humans and mice.

Identification of human cytotoxic ILC3s.

Human ILCs are classically categorized into five subsets; cytotoxic CD127- CD94+ NK cells and non-cytotoxic CD127+ CD94- , ILC1s, ILC2s, ILC3s, and LTi cells. Here, we identify a previously unrecognized subset within the CD127+ ILC population, characterized by the expression of the cytotoxic marker CD94. These CD94+ ILCs resemble conventional ILC3s in terms of phenotype, transcriptome, and cytokine production, but are highly cytotoxic. IL-15 was unable to induce differentiation of CD94+ ILCs toward mature NK cells. Instead, CD94+ ILCs retained RORγt, CD127 and CD200R1 expression and produced IL-22 in response to IL-15. Culturing non-cytotoxic ILC3s with IL-12 induced upregulation of CD94 and cytotoxic activity, effects that were not observed with IL-15 stimulation. Thus, human helper ILCs can acquire a cytotoxic program without differentiating into NK cells.

Author Correction: c-Kit-positive ILC2s exhibit an ILC3-like signature that may contribute to IL-17-mediated pathologies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human ectoenzyme-expressing ILC3: immunosuppressive innate cells that are depleted in graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often associated with chemotherapy- and radiotherapy-induced host tissue damage, leading to graft-versus-host disease (GVHD). Innate lymphoid cells (ILC) have an essential role in tissue homeostasis and tissue repair via their production of interleukin (IL)-22, which acts on intestinal stem cells. The tissue healing capacities of ILC via IL-22 in the context of allo-HSCT and GVHD has previously been demonstrated in a mouse model for acute GVHD. We investigated potential other ways of ILC-mediated tissue protection against GVHD. Tissue injury leads to the release of danger-associated molecular patterns (DAMPs). DAMPs interact with purinergic receptors and ectoenzymes on immune cells and induce pleiotropic effects, including activation of proinflammatory antigen-presenting cells and immunosuppressive effects via the generation of adenosine. Here, we report a novel subset of human ILC3 that coexpress the ectoenzymes CD39 and CD73 (ecto+ ILC3). Ecto+ ILC3 express RORγt and were present in the oral-gastrointestinal tract and bone marrow. ILC3 ectoenzyme expression is modulated by the proinflammatory cytokine IL-1ß. Extracellular adenosine triphosphate (eATP) stimulated ecto+ ILC3 to produce IL-22 and adenosine. Activated ecto+ ILC3 suppressed autologous T-cell proliferation in coculture experiments via the production of adenosine. In allo-HSCT recipients, intestinal GVHD was associated with reduced proportions of ecto+ ILC3 and decreased levels of adenosine and its metabolite inosine. Taken together, ecto+ ILC3 have immunosuppressive properties, but in patients with GVHD, ecto+ ILC3 are depleted. A lack of ecto+ ILC3 and subsequent reduced capacity to neutralize DAMPs may contribute to the development of GVHD.

c-Kit-positive ILC2s exhibit an ILC3-like signature that may contribute to IL-17-mediated pathologies.

Here we identify a group 2 innate lymphoid cell (ILC2) subpopulation that can convert into interleukin-17 (IL-17)-producing NKp44- ILC3-like cells. c-Kit and CCR6 define this ILC2 subpopulation that exhibits ILC3 features, including RORγt, enabling the conversion into IL-17-producing cells in response to IL-1ß and IL-23. We also report a role for transforming growth factor-ß in promoting the conversion of c-Kit- ILC2s into RORγt-expressing cells by inducing the upregulation of IL23R, CCR6 and KIT messenger RNA in these cells. This switch was dependent on RORγt and the downregulation of GATA-3. IL-4 was able to reverse this event, supporting a role for this cytokine in maintaining ILC2 identity. Notably, this plasticity has physiological relevance because a subset of RORγt+ ILC2s express the skin-homing receptor CCR10, and the frequencies of IL-17-producing ILC3s are increased at the expense of ILC2s within the lesional skin of patients with psoriasis.

KLRG1 and NKp46 discriminate subpopulations of human CD117+CRTH2- ILCs biased toward ILC2 or ILC3.

Recently, human ILCs that express CD117 and CD127 but lack CRTH2 and NKp44 have been shown to contain precursors of ILC1, ILC2, and ILC3. However, these ILCs have not been extensively characterized. We performed an unbiased hierarchical stochastic neighbor embedding (HSNE) analysis of the phenotype of peripheral blood CD117+ ILCs, which revealed the presence of three major subsets: the first expressed NKp46, the second expressed both NKp46 and CD56, and the third expressed KLRG1, but not NKp46 or CD56. Analysis of their cytokine production profiles and transcriptome revealed that NKp46+ ILCs predominantly develop into ILC3s; some of them can differentiate into ILC1/NK-like cells, but they are unable to develop into ILC2s. In contrast, KLRG1+ ILCs predominantly differentiate into ILC2s. Single-cell cultures demonstrate that KLRG1+ ILCs can also differentiate into other ILC subsets depending on the signals they receive. Epigenetic profiling of KLRG1+ ILCs is consistent with the broad differentiation potential of these cells.

Antibody opsonization enhances MAIT cell responsiveness to bacteria via a TNF-dependent mechanism.

Mucosal-associated invariant T (MAIT) cells are an abundant human T-cell subset with antimicrobial properties. They can respond to bacteria presented via antigen-presenting cells (APCs) such as macrophages, which present bacterially derived ligands from the riboflavin synthesis pathway on MR1. Moreover, MAIT cells are also highly responsive to cytokines which enhance and even substitute for T-cell receptor-mediated signaling. The mechanisms leading to an efficient presentation of bacteria to MAIT cells by APCs have not been fully elucidated. Here, we showed that the monocytic cell line THP-1 and B cells activated MAIT cells differentially in response to Escherichia coli. THP-1 cells were generally more potent in inducing IFNγ and IFNγ/TNF production by MAIT cells. Furthermore, THP-1, but not B, cells produced TNF upon bacterial stimulation, which in turn supported IFNγ production by MAIT cells. Finally, we addressed the role of antibody-dependent opsonization of bacteria in the activation of MAIT cells using in vitro models. We found that opsonization had a substantial impact on downstream MAIT cell activation by monocytes. This was associated with enhanced activation of monocytes and increased TNF release. Importantly, this TNF acted in concert with other cytokines to drive MAIT cell activation. These data indicate both a significant interaction between adaptive and innate immunity in the response to bacteria, and an important role for TNF in MAIT cell triggering.

New insights into the function, development, and plasticity of type 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2s) are the most well defined group of ILCs. ILC2 development is controlled by the GATA-3 transcription factor and these cells produce archetypal type 2 cytokines, such as IL-5 and IL-13. These cytokines mediate parasite expulsion and tissue repair, but also contribute to type 2 inflammatory diseases, including allergy, asthma and chronic rhinosinusitis with nasal polyps. In response to tightly regulated local environmental cues ILCs can generate characteristics of other subtypes, a process known as plasticity. Recent advances in the ILC2 field has led to the discovery that ILC2s can promptly shift to functional IFN-γ-producing ILC1s or IL-17-producing ILC3s, depending on the cytokines and chemokines produced by antigen presenting cells or epithelial cells. Due to yet unknown triggers, this complex network of signals may become dysregulated. In this review, we will discuss general ILC characteristic, ILC2 development, plasticity, memory function, and implications in disease.

Isolation of Human Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8+ T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc.

FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming.

CD103+ dendritic cells (DC) are crucial for regulation of intestinal tolerance in humans. However, upon infection of the lamina propria this tolerogenic response is converted to an inflammatory response. Here we show that immunoglobulin A (IgA) immune complexes (IgA-IC), which are present after bacterial infection of the lamina propria, are important for the induction of inflammation by the human CD103+SIRPα+ DC subset. IgA-IC, by recognition through FcαRI, selectively amplify the production of proinflammatory cytokines TNF, IL-1ß and IL-23 by human CD103+ DCs. These cells then enhance inflammation by promoting Th17 responses and activating human intestinal innate lymphoid cells 3. Moreover, FcαRI-induced cytokine production is orchestrated via upregulation of cytokine translation and caspase-1 activation, which is dependent on glycolytic reprogramming mediated by kinases Syk, PI3K and TBK1-IKKε. Our data suggest that the formation of IgA-IC in the human intestine provides an environmental cue for the conversion of a tolerogenic to an inflammatory response.

Interleukin-12 and -23 Control Plasticity of CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria.

Human group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127(+) ILC1, and intraepithelial CD103(+) ILC1. In inflamed intestinal tissues from Crohn's disease patients, numbers of CD127(+) ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127(+) ILC1 is reversible in vitro and in vivo. CD127(+) ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1ß dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohn's disease patients a higher proportion of CD14(+) dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127(+) ILC1. In contrast, CD14(-) DCs promoted differentiation from CD127(+) ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127(+) ILC1 and ILC3 in the gut.