Total Innate Lymphoid Cells Research Articles

Histology of the Upper Gastrointestinal Tract, Morphometry and Lymphocyte Subpopulations of the Duodenal Mucosa: Insights from Healthy Individuals

The upper oesophagogastrointestinal (UEGI) tract histology, intestinal morphometry and lymphocyte subpopulations of healthy people is scarcely known. In research studies of inflammation involving the UEGI tract, there is a lack of adequate healthy controls. Aims: To evaluate the histology of the UEGI tract and the duodenal lymphocyte subpopulations of healthy volunteers and patients with gastroesophageal reflux disease (GERD), the latter to assess if it could replace healthy subjects. Healthy individuals were excluded if they had symptoms, comorbidities, pregnancy, toxics, medications or abnormal blood analysis. Subjects in both groups with abnormal duodenal intraepithelial lymphocyte (IEL) counts were also excluded. A total of 280 subjects were assessed, and 37 were included (23 healthy and 14 with GERD). The GERD group showed a higher IEL count (median [IQR]: 19.5 [17–22]) than healthy group: (15 [12–18]), p = 0.004. Eosinophils, mast cells and intestinal morphometry were similar in both groups. In the lamina propria, CD4+ T cells decreased (p = 0.008), and CD8+ T cells increased (p = 0.014). The total innate lymphoid cells (ILC) and CD3− cells decreased (p = 0.007) in GERD group compared to healthy controls. At the intraepithelial level, NKT cells increased (p = 0.036) and ILC3 decreased (p = 0.049) in the GERD group. This is the first study to comprehensively map the histology, morphometry and duodenal subpopulations of healthy volunteers to help define a “gold standard” of normality. The differences found between both groups suggest that, whenever possible, healthy subjects should be included in research studies. Alternatively, we can consider a well-defined homogenous group with GERD to serve as the control group.

The Tipped Balance of ILC1/ILC2 in Peripheral Blood of Oral Lichen Planus Is Related to Inflammatory Cytokines.

Oral lichen planus (OLP) is an immune-inflammatory disease mediated by T cells. Innate lymphoid cells (ILCs) constitute a novel family of immune cells that initially originate from common innate lymphoid progenitors. Termed “T cells counterparts,” ILCs play a prominent role in inflammatory-immune diseases. However, the characterization of ILCs and their related induced factors were unclear in OLP. In the present study, the phenotypic characteristics of ILCs and their correlation with inflammatory cytokines were explored in the peripheral blood of OLP patients and healthy controls. We found that the proportion of total ILCs was expanded in OLP and was positively correlated with disease severity. The highly skewed distribution of ILC subpopulations was notable in OLP. Specifically, the frequency of ILC1s was significantly increased, while that of ILC2s was significantly reduced in total ILCs of OLP, resulting in the markedly elevated ILC1/ILC2 ratio in OLP. Correspondingly, ILCs in OLP displayed high expression of T-bet but low expression of GATA3. In addition, the IFN-γ expression level was elevated in ILC1s, whereas the IL-4 expression level was decreased in ILC2s. Moreover, ILC-associated activators IL-12, IL-18, and IL-1β were upregulated in OLP plasma, with IL-12 and IL-1β both positively correlated with the ILC1/ILC2 ratio. Further in vitro stimulation tests indicated that OLP plasma remarkedly increased the ILC1/ILC2 ratio, especially that IL-12 and IL-1β tipped the balance between ILC1s and ILC2s toward ILC1s in total ILCs. Overall, elevated levels of IL-12 and IL-1β might act as environmental cues in tipping the balance of ILC1/ILC2 in the peripheral blood of OLP, contributing to the immune dysregulation in OLP.

Circulating Innate Lymphoid Cells Exhibit Distinctive Distribution During Normal Pregnancy

Over the past decades, the investigation of innate lymphoid cells (ILCs) has revealed their significance in successful pregnancy. Sex hormones, such as estradiol and progesterone, show specific changes during pregnancy and modulate both adaptive and innate immune systems. ILC subset distribution in peripheral blood of pregnant women and its potential association with sex hormone levels have not been well revealed. Peripheral blood was obtained from healthy non-pregnant, early-pregnant, and late-pregnant women. Radioimmunoassay was performed to measure plasma estradiol and progesterone levels. The levels of type 1 ILCs (ILC1s), type 2 ILCs (ILC2s), type 3 ILCs (ILC3s), and total ILCs as well as estrogen and progesterone receptors of ILC2s in peripheral blood were analyzed using flow cytometry. The proportion of total ILCs and distribution of ILC subsets in peripheral blood changed dynamically during pregnancy. Compared to non-pregnant women, late-pregnant women displayed significantly higher proportion of circulating ILCs, among which ILC2s accounted for the majority in late-pregnant women while a smaller part in others, and ILC3s displayed the opposite. Plasma estradiol and progesterone levels elevated while pregnancy proceeded and the expression of their receptors in ILC2s increased consisted with the proportion of circulating ILC2s. Our work first observed the existence of progesterone receptors in human circulating ILC2s and revealed the distribution pattern of circulating ILC subsets and their interrelation with plasma sex hormone levels during pregnancy. Our results suggested that the estradiol and progesterone levels might partly influence the distribution of circulating ILC subsets and implied the interplay between circulating ILCs and pregnancy.

Therapeutic effects of vitamin D and IL-22 on methotrexate-induced mucositis in mice.

Mucositis is a common side effect of cancer therapies and transplant conditioning regimens. Management of mucositis involves multiple approaches from oral hygiene, anti-inflammatory, anti-apoptotic, cytoprotective, and antioxidant agents, to cryo-therapy, physical therapy, and growth factors. There is room for novel, affordable treatment options, or improvement of currently available therapies. Vitamin D has been shown to regulate mucosa-resident cell populations such as Th17 or innate lymphoid cells and critical mucosal cytokine IL-22; however, their therapeutic potential has not been put to test in preclinical mouse models. In this study, we aimed to test the therapeutic potential of vitamin D injections and IL-22 overexpression in a murine model of chemotherapy-induced mucositis. Balb/c mice were given daily intraperitoneal injections of vitamin D. Mucositis was induced by methotrexate. Another group received IL-22 plasmid via hydrodynamic gene delivery. Weight loss and intestinal histopathology, intestinal levels of cytokines IL-22, IL-17A, GM-CSF, IL-23, IFN-γ, TNF-α, and IL-10, and number of intestinal lamina propria B cell, neutrophil, and total innate lymphoid cells were quantified. Daily vitamin D injections ameliorated intestinal inflammation and elevated intestinal IL-22 levels compared with control groups. Temporal overexpression of IL-22 by hydrodynamic gene delivery slightly increased intestinal IL-22 but failed to confer significant protection from mucositis. To our knowledge, this is the first experimental demonstration in an animal model of mucositis of therapeutic use of vitamin D and IL-22 supplementation and our results with vitamin D suggest it may have merit in further trials in human mucositis patients.

ILC1s and ILC3s Exhibit Inflammatory Phenotype in Periodontal Ligament of Periodontitis Patients.

Innate lymphoid cells (ILCs) are emerging as important players in inflammatory diseases. The oral mucosal barrier harbors all ILC subsets, but how these cells regulate the immune responses in periodontal ligament tissue during periodontitis remains undefined. Here, we show that total ILCs are markedly increased in periodontal ligament of periodontitis patients compared with healthy controls. Among them, ILC1s and ILC3s, particularly NKp44+ILC3 subset, are the predominant subsets accumulated in the periodontal ligament. Remarkably, ILC1s and ILC3s from periodontitis patients produce more IL-17A and IFN-γ than that from healthy controls. Collectively, our results highlight the role of ILCs in regulating oral immunity and periodontal ligament inflammation and provide insights into targeting ILCs for the treatment of periodontitis.

Inflammatory status might direct ILC and NK cells to IL-17 expressing ILC3 and NK subsets in Behcet's disease

Innate lymphoid cells (ILCs) are lymphoid cells that have important effector and regulatory functions in innate immunity and tissue remodeling. Uncontrolled activation and proliferation of ILCs can contribute to inflammatory autoimmune diseases. Behcet's disease (BD) is a complex systemic inflammatory disorder of unknown etiology. It has been shown that natural killer (NK) cells may play an immunoregulatory role in BD, however the role of ILCs is unknown. In this study, the levels and functions of ILCs and NK cell subsets in BD patients were investigated. Cell surface and cytotoxic granules (perforin and granzyme) expression of NK cells and ILCs were evaluated and labeled according to whole blood lysing protocol in peripheral blood samples obtained from the patients and healthy subjects. Cytokine levels of NK cells were investigated in stimulated peripheral blood mononuclear cells. All data were analyzed by flow cytometry. Total ILC and ILC3+ cells were increased in active BD patients compared to inactive BD patients and healthy subjects. There was no significant difference between the patients and healthy subjects regarding NK cell surface and intracellular molecule expression. Although, an increase in IFN-γ and IL-17, and a decrease in IL-4 levels were observed in CD56dim NK cell subset of BD patients. Recent studies showed increased neutrophilic infiltration and IL-17 secreting Th17 cells in BD patients. It is known that ILC3+cells are similar to Th17 subset regarding their cytokine profile and transcription factor expression patterns. Results of current study may suggest that inflammatory microenvironment in BD patients might direct ILC cells to differentiate into ILC3+ subset, and IL-17 released by NK cells might have a role in neutrophilic infiltration.

Abstract 3310: Resolving the heterogeneity of human circulating innate lymphoid cells via simultaneous, high-dimensional analysis of protein and gene expression

Abstract 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.

Resolving the heterogeneity of human circulating innate lymphoid cells via simultaneous, high-dimensional analysis of protein and gene expression

Abstract Cancer treatment has been revolutionized with the development of immunomodulatory therapies. While these therapies have primarily focused on enhancing T-cell responses, there has been interest in harnessing the 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. However, there is a need for better understanding of these recently described cells. In this study, we developed a comprehensive approach to further refine the signatures of human circulating ILC subsets. Total ILCs were enriched 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 enabled 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 3 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. ILC subsets differentially distributed across donors were detected and defined using unsupervised computational analysis confirming the result of multiomic analysis. For Research Use Only. Class 1 laser product. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Imbalance of Circulating Innate Lymphoid Cell Subpopulations in Patients With Septic Shock.

Background: Septic shock, a major cause of death in critical care, is the clinical translation of a cytokine storm in response to infection. It can be complicated by sepsis-induced immunosuppression, exemplified by blood lymphopenia, an excess of circulating Treg lymphocytes, and decreased HLA-DR expression on circulating monocytes. Such immunosuppression is associated with secondary infections, and higher mortality. The effect of these biological modifications on circulating innate lymphoid cells (ILCs) has been little studied.Methods: We prospectively enrolled patients with septic shock (Sepsis-3 definition) in the intensive care unit (ICU) of Timone CHU Hospital. ICU controls (trauma, cardiac arrest, neurological dysfunction) were recruited at the same time (NCT03297203). We performed immunophenotyping of adaptive lymphocytes (CD3+ T cells, CD19+ B cells, CD4+CD25+FoxP3+ Treg lymphocytes), ILCs (CD3−CD56+ NK cells and helper ILCs – ILC1, ILC2, and ILC3), and monocytes by flow cytometry on fresh blood samples collected between 24 and 72 h after admission.Results: We investigated adaptive and innate circulating lymphoid cells in the peripheral blood of 18 patients in septic shock, 15 ICU controls, and 30 healthy subjects. As expected, the peripheral blood lymphocytes of all ICU patients showed lymphopenia, which was not specific to sepsis, whereas those of the healthy volunteers did not. Circulating CD3+ T cells and CD3−CD56+ NK cells were mainly concerned. There was a tendency toward fewer Treg lymphocytes and lower HLA-DR expression on monocytes in ICU patients with sepsis. Although the ILC1 count was higher in septic patients than healthy subjects, ILC2, and ILC3 counts were lower in both ICU groups. However, ILC3s within the total ILCs were overrepresented in patients with septic shock. The depression of immune responses has been correlated with the occurrence of secondary infections. We did not find any differences in ILC distribution according to this criterion.Conclusion: All ICU patients exhibit lymphopenia, regardless of the nature (septic or sterile) of the initial medical condition. Specific distribution of circulating ILCs, with an excess of ILC1, and a lack of ILC3, may characterize septic shock during the first 3 days of the disease.

MAIT cells, TCR γδ+ cells and ILCs cells in human breast milk and blood from HIV infected and uninfected women.

Human breast milk cells remain poorly characterized for the presence of unconventional T lymphocytes and innate lymphoid cells (ILCs). Early breast milk was collected from eight HIV-uninfected and 11 HIV-infected women 3-12days after delivery. Mucosal-associatedinvariant T cells (MAIT cells), TCR γδ cells, and innate lymphoid cells (ILCs) were analyzed in breast milk and paired blood samples. CD161+/TRAV1-2+MAIT cells were detected in breast milk, accounting for a median (IQR) of 0.08% (0.06-0.16) and 0.17% (0.16-0.31) of CD45+ breast milk cells in HIV-uninfected and HIV-infected women, respectively. A selective compartmentalization of γδ T lymphocytes was observed in breast milk. Median (IQR) frequency of γδ T lymphocytes was 8.95% (8.64-12.14) among breast milk lymphocyte cells compared to 2.54% (1.81-4.10) in blood (P=0.03) in HIV-uninfected women, and 7.26% (4.22-10.54) in breast milk versus 3.31% (2.54-3.80) in blood (P=0.004) from HIV-infected women. The proportion of group 1 ILC (ILC1) among total ILCs was higher in breast milk compared to blood in HIV-uninfected women (P=0.03) and HIV-infected women (P=0.001). The frequency of ILC2 among total ILCs tends to be lower in breast milk compared to blood in HIV-uninfected women (P=0.06) and HIV-infected women (P=0.03). Unconventional T cells and ILCs that may be involved in both the protection against infection of the lactating mammary gland and maturation of infant's gut and microbiomes account for a detectable fraction of breast milk cells.

Preferential Reduction of Circulating Innate Lymphoid Cells Type 2 in Patients with Common Variable Immunodeficiency with Secondary Complications Is Part of a Broader Immune Dysregulation

Over a third of patients with common variable immunodeficiency (CVID) suffer from secondary complications like inflammatory organ disease, autoimmune manifestations, or lymphoproliferation contributing to increased morbidity and mortality in affected patients. Innate lymphoid cells (ILCs) have emerging roles in setting the milieu for physiological, but also pathological, immune responses and inflammation. We therefore sought to correlate the recently identified disturbed homeostasis of ILCs with alterations of the adaptive immune system in complex CVID patients (CVIDc). We quantified peripheral blood ILC and T helper cell subsets of 58 CVID patients by flow cytometry and compared the results to the clinical and immunological phenotype. Total ILCs were significantly reduced in peripheral blood of CVIDc patients compared to healthy individuals, but not to CVID patients who suffered only from infections (CVIDio). This reduction was mainly due to a decrease in ILC2s, while ILC3s were relatively increased in CVIDc compared to CVIDio patients. This alteration in ILC phenotype was more prominent in patients with an expansion of CD21low B cells, but we could not detect an association of the altered ILC phenotype with a TH1-shift among circulating CD4 T cells, which was also prominent in CVIDc patients. We confirm a relative shift in ILCs of CVIDc patients towards ILC3s which was associated with the expansion of CD21low B cells, but not overtly with the relative expansion of TH1-like T cells. Given the relative abundance of TH1-like T cells compared to ILCs, these probably represent a more prominent source of the observed IFNγ-signature in CVIDc patients.

Circulating innate lymphoid cells are unchanged in response to DAC HYP therapy

Innate lymphoid cells (ILCs) play an important role in immunity, inflammation, and tissue remodeling and their dysregulation is implicated in autoimmune and inflammatory disorders. We analyzed the impact of daclizumab, a humanized monoclonal anti-CD25 antibody, on circulating natural killer (NK) cells and ILCs in a cohort of multiple sclerosis patients. An increase in CD56(bright) NK cells and CD56(hi)CD16(intermediate) transitional NK cells was observed. No significant change in total ILCs or major ILC subpopulations was observed. These results refine our understanding of the impact of daclizumab on innate lymphoid cell populations.

OC.12.4 INNATE LYMPHOID CELLS ARE INCREASED IN CELIAC DISEASE INTESTINAL MUCOSA AND HAVE A PATHOGENIC ROLE IN A MOUSE MODEL OF SMALL INTESTINE ATROPHY

Background and aim: Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of our study was to characterize the tissue distribution of ILCs in celiac disease (CD) and analyze their role in gut tissue damage. Materials and methods: ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs), isolated from duodenal biopsies of patients with active CD, patients with inactive CD on a gluten-free diet and healthy controls (HC) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines, transcription factors and receptors for interleukin (IL)-15, interferon (IFN)-alpha and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with IL-15, IFNalpha and poly I:C, a TLR agonist, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Results: The percentage of total ILCs did not differ between CD (both active and inactive) patients and HC. However, the fractions of ILCs type 1 expressing T-bet and ILCs type 3 expressing ROR-(g)t were significantly increased in active CD compared to controls, while there was no difference in term of ILCs type 2 among groups. ILCs expressed TLR3, IL-15R, little TLR2 but not IFN-alphaR and TLR4. IL-15 and poly I:C, but not IFN-alpha increased IFNgamma expression in ILCs. In vivo in mice, depletion of ILCs significantly reduced TLR3driven small intestinal damage. Conclusions: Active CD is characterized by preferential accumulation of type 1 and type 3 ILC subpopulations and these cells respond to IL-15, a cytokine over-produced in CD, by enhancing IFN-gamma production. The demonstration that ILCs express TLR3, are functionally able to respond to poly I:C with increased synthesis of inflammatory cytokines, and contribute to TLR3-driven small intestinal atrophy delineates a novel mechanism underlying the CD-associated tissue damage.