ILC Subsets Research Articles

HVEM expression by innate lymphoid cells protects against enteric bacterial infection

Abstract Yersinia enterocolitica causes food-borne disease and targets the small intestine, in which ILC3 are the predominant ILC subset. Here we show that ILC3 are required for protection of mice from oral Y. enterocolitica, as mice lacking ILC3 were more susceptible and transfer of ILC to recipients was protective. IFNγ is a cytokine required for host defense from this pathogen, and while ILC in the small intestine did not increase in number they greatly increased IFNγ production after infection. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily (TNFRSF), is expressed by all intestinal ILC subsets. Mice with a deficiency of HVEM expression in RORγt+ ILC had a reduced steady-state IL-22 production, and in vitro HVEM could signal to ILC3 to stimulate IL-22 secretion. Moreover, following oral infection with Y. enterocolitica, mice with HVEM deficiency mediated by Rorc-cre showed reduced survival and increased bacterial translocation early after infection. Cytokines are known to induce ILC activation, but our results demonstrate a novel role for the cell surface receptor HVEM in regulating ILC3 cytokine production, both at steady state, and as a critical survival factor during acute infection.

Partially Maintaining E Protein Activity During ILC Development Selectively Inhibits ILC2 Development

Abstract Innate lymphoid cells (ILCs) provide an early response to pathogenic damage at mucosal barrier surfaces. They have beneficial roles in infection and tissue repair, as well as pathogenic roles in chronic inflammatory diseases. ILC effector subsets are defined by transcription factor expression patterns and cytokine production profiles. Recent evidence suggests ILC cytokine profiles are not static and cells can be pushed into producing different cytokines. The possibility of manipulating ILCs toward beneficial immune responses or away from pathogenic responses increases the urgency to understand how ILC effector programs are established during ILC development and maintained during homeostasis and immune responses. All ILCs are generated from a common precursor derived from the common lymphoid progenitor, and differentiation of this precursor into ILC1s, ILC2s, and ILC3s is characterized by induction of distinct transcription factors. All ILC subsets share a developmental requirement for Id2 induction. Id2 is an inhibitor of E protein transcription factor activity, but its specific functions in ILC development have not been elucidated. We have used expression of ET-2, a chimeric protein that acts as an Id dominant negative inhibitor, to partially block Id2 function during ILC development. Our experiments show that while ILC1s and ILC3s are mostly undisturbed by ET-2, ILC2s experience a profound block in development. These results demonstrate that, while all ILCs require Id2 expression, ILC2s are uniquely sensitive to the effects of ET-2 and require a more complete inhibition of E protein activity.

Dysregulation of Group 3 Innate Lymphoid Cells in the Pathogenesis of Inflammatory Bowel Disease.

Purpose of ReviewHere, we review recent literature indicating a role of innate lymphoid cells in human inflammatory bowel disease with a focus on the plastic population of ILC3.Recent FindingsMany studies suggest an involvement of ILC3 in human intestinal inflammation. ILC3 present the most abundant ILC subtype in the human intestine at steady state. In IBD, this composition is skewed towards ILCs showing an ILC1 phenotype and cytokine profile. This change is likely due to the microenvironment causing skewing of the functionally plastic ILC subsets. Interactions between ILCs and other cells are important to keep homeostasis and intestinal barrier integrity.SummaryThe knowledge about the involvement of ILCs in IBD is rapidly increasing, and with the help of mouse models, new pathways and functions of ILCs are continuously unraveled. In the majority of human studies, a potential role for ILCs in Crohn’s disease is found. However, less data is available for a possible role in ulcerative colitis. Results from mice are obtained from diverse model systems, and more research in this field is needed to clarify and integrate the current knowledge in order to improve treatment strategies for IBD patients.

Murine liver-resident group 1 innate lymphoid cells regulate optimal priming of anti-viral CD8+ T cells.

The liver contains 2 transcriptionally distinct group 1 ILC subsets: CD49a+ ILC1s and CD49b+ NK cells. However, little is known about how group 1 ILCs contribute to hepatic immune responses. Therefore, we characterized murine liver-resident group 1 ILCs and found that CD49a+ ILC1s express high levels of the inhibitory receptor NKG2A and localize near DCs in perivascular spaces surrounding the portal triads. Upon hepatic viral infection, NKG2A signaling in group 1 ILCs, especially in CD49a+ ILC1s, inhibits CXCL9 expression required for robust accumulation of IFN-γ+CD49b+ NK cells. As a consequence, NKG2A-/- mice showed increased numbers of IFN-γ-producing NK cells that preferentially activate liver CD103+ DCs, leading to the sustained proliferation of adoptively transferred, virus-specific CD8+ T cells. Collectively, these data suggest that group 1 ILCs play a role in maintaining the liver as a tolerogenic site by limiting the recruitment of peripheral NK cells during the early phase of viral infection. Furthermore, our findings implicate that the inhibition of NKG2A signaling on group 1 ILCs may be a novel vaccine strategy to induce robust CD8+ T cell responses against persistent liver pathogens.

Ikaros Inhibits Group 3 Innate Lymphoid Cell Development and Function by Suppressing the Aryl Hydrocarbon Receptor Pathway

Group 3 innate lymphoid cells (ILC3s) expressing the transcription factor (TF) RORγt are important for the defense and homeostasis of host intestinal tissues. The zinc finger TF Ikaros, encoded by Ikzf1, is essential for the development of RORγt(+) fetal lymphoid tissue inducer (LTi) cells and lymphoid organogenesis, but its role in postnatal ILC3s is unknown. Here, we show that small-intestinal ILC3s had lower Ikaros expression than ILC precursors and other ILC subsets. Ikaros inhibited ILC3s in a cell-intrinsic manner through zinc-finger-dependent inhibition of transcriptional activity of the aryl hydrocarbon receptor, a key regulator of ILC3 maintenance and function. Ablation of Ikzf1 in RORγt(+) ILC3s resulted in increased expansion and cytokine production of intestinal ILC3s and protection against infection and colitis. Therefore, in contrast to being required for LTi development, Ikaros inhibits postnatal ILC3 development and function to regulate gut immune responses at steady state and in disease.

C-type lectin-like NKC-encoded immunoreceptors NKp80 and NKp65 are selectively expressed by human innate lymphocyte subsets, uniquely signal via hemITAMs and facilitate tissue-specific immunosurveillance via their genetically linked ligands AICL and KACL

Abstract NKp80 is an activating C-type lectin-like immunoreceptor (CTLR) on human NK cells. Upon ligation by its cognate ligand ‘activation-induced C-type lectin’ (AICL) up-regulated on PAMP-stimulated myeloid cells and interleukin-experienced NK cells, NKp80 stimulates NK cytotoxicity and cytokine secretion. Unlike other NK receptors, NKp80 uses an atypical cytoplasmic hemITAM for cellular activation. In contrast to “classical” hemITAMs of related myeloid CTLR, we showed that the NKp80 hemITAM does not recruit Syk upon phosphorylation. However, we now provide evidence for specific recruitment of other signaling molecules by NKp80. Alike its close relative NKp80, NKp65 is a homodimeric CTLR with a cytoplasmic hemITAM and encoded in the Natural Killer Gene Complex (NKC). NKp65 binds with high affinity to its cognate genetically linked ligand KACL almost exclusively expressed by human keratinocytes. We previously showed that NKp65/KACL interaction activates cytotoxicity by NK-92 cells. However, while NKp80 is expressed by virtually all human NK cells, physiological expression of NKp65 remained elusive. Generation of NKp65-specific mAb enabled us to trace NKp65 expression to a minor subset of human innate lymphocytes. Transcriptomics revealed strict correlation between NKp65 expression ILC hallmark genes. Hence, NKp65 expression defines that ILC subset and enables a tissue-specific cross-talk with human keratinocytes. Moreover, we show that NKp80 and NKp65 are mutually exclusive expressed by NK cells and that ILC subset, qualifying NKp80 and NKp65 as markers of these 2 distinct innate lymphocyte lineages and raising questions for their respective subset-associated function.

The ETS1 transcription factor is required for the development and cytokine-induced expansion of ILC2.

Group 2 innate lymphoid cells (ILC2s) are a subset of ILCs that play a protective role in the response to helminth infection, but they also contribute to allergic lung inflammation. Here, we report that the deletion of the ETS1 transcription factor in lymphoid cells resulted in a loss of ILC2s in the bone marrow and lymph nodes and that ETS1 promotes the fitness of the common progenitor of all ILCs. ETS1-deficient ILC2 progenitors failed to up-regulate messenger RNA for the E protein transcription factor inhibitor ID2, a critical factor for ILCs, and these cells were unable to expand in cytokine-driven in vitro cultures. In vivo, ETS1 was required for the IL-33-induced accumulation of lung ILC2s and for the production of the T helper type 2 cytokines IL-5 and IL-13. IL-25 also failed to elicit an expansion of inflammatory ILC2s when these cells lacked ETS1. Our data reveal ETS1 as a critical regulator of ILC2 expansion and cytokine production and implicate ETS1 in the regulation of Id2 at the inception of ILC2 development.

Characterization and Quantification of Innate Lymphoid Cell Subsets in Human Lung.

BackgroundInnate lymphoid cells (ILC) are a new family of innate immune cells that have emerged as important regulators of tissue homeostasis and inflammation. However, limited data are available concerning the relative abundance and characteristics of ILC in the human lung.MethodsThe aim of this study was to characterize and enumerate the different ILC subsets in human lung by multi-color flow cytometry.ResultsWithin the CD45+ Lin- CD127+ pulmonary ILC population, we identified group 1 (ILC1), group 2 (ILC2) and group 3 (ILC3) innate lymphoid cells using specific surface markers (i.e. IL12Rβ2, CRTH2 and CD117 respectively) and key transcription factors (i.e. T-bet, GATA-3 and RORγT respectively). Based on the presence of NKp44, ILC3 were further subdivided in natural cytotoxicity receptor (NCR)+ and NCR- ILC3. In addition, we demonstrated the production of signature cytokines IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF in the pulmonary ILC population. Interestingly, we observed a tendency to a higher frequency of NCR- ILC3 in lungs of patients with chronic obstructive pulmonary disease (COPD) compared with controls.ConclusionsWe show that the three main ILC subsets are present in human lung. Importantly, the relative abundance of ILC subsets tended to change in COPD patients in comparison to control individuals.

Innate Lymphoid Cells Groups 1 and 3 in the Epithelial Compartment of Functional Human Intestinal Allografts

We examined intraepithelial lymphocytes (IELs) in 213 ileal biopsies from 16 bowel grafts and compared them with 32 biopsies from native intestines. During the first year posttransplantation, grafts exhibited low levels of IELs (percentage of CD103(+) cells) principally due to reduced CD3(+) CD8(+) cells, while CD103(+) CD3(-) cell numbers became significantly higher. Changes in IEL subsets did not correlate with histology results, isolated intestine, or multivisceral transplants, but CD3(-) IELs were significantly higher in patients receiving corticosteroids. Compared with controls, more CD3(-) IELs of the grafts expressed CD56, NKp44, interleukin (IL)-23 receptor, retinoid-related orphan receptor gamma t (RORγt), and CCR6. No difference was observed in granzyme B, and CD3(-) CD127(+) cells were more abundant in native intestines. Ex vivo, and after in vitro activation, CD3(-) IELs in grafts produced significantly more interferon (IFN)-γ and IL-22, and a double IFNγ(+) IL-22(+) population was observed. Epithelial cell-depleted grafts IELs were cytotoxic, whereas this was not observed in controls. In conclusion, different from native intestines, a CD3(-) IEL subset predominates in grafts, showing features of natural killer cells and intraepithelial ILC1 (CD56(+) , NKp44(+) , CCR6(+) , CD127(-) , cytotoxicity, and IFNγ secretion), ILC3 (CD56(+) , NKp44(+) , IL-23R(+) , CCR6(+) , RORγt(+) , and IL-22 secretion), and intermediate ILC1-ILC3 phenotypes (IFNγ(+) IL-22(+) ). Viability of intestinal grafts may depend on the balance among proinflammatory and homeostatic roles of ILC subsets.

Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation

Background & AimsInnate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation.MethodsILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota.ResultsIL17A- and IL22-producing, natural cytotoxicity receptor–negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor–positive cells, in a dose-dependent manner.ConclusionsIL6 contributes to activation of colonic natural cytotoxicity receptor–negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor–positive cells.

CXCR6 Expression Is Important for Retention and Circulation of ILC Precursors.

Innate lymphoid cells are present at mucosal sites and represent the first immune barrier against infections, but what contributes to their circulation and homing is still unclear. Using Rag2 −/− Cxcr6 Gfp/+ reporter mice, we assessed the expression and role of CXCR6 in the circulation of ILC precursors and their progeny. We identify CXCR6 expressing ILC precursors in the bone marrow and characterize their significant increase in CXCR6-deficient mice at steady state, indicating their partial retention in the bone marrow after CXCR6 ablation. Circulation was also impaired during embryonic life as fetal liver from CXCR6-deficient embryos displayed decreased numbers of ILC3 precursors. When injected, fetal CXCR6-deficient ILC3 precursors also fail to home and reconstitute ILC compartments in vivo. We show that adult intestinal ILC subsets have heterogeneous expression pattern of CXCR6, integrin α 4 β 7, CD62L, CD69, and CD44, with ILC1 and ILC3 being more likely tissue resident lymphocytes. Intestinal ILC subsets were unchanged in percentages and numbers in both mice. We demonstrate that the ILC frequency is maintained due to a significant increase of ILC peripheral proliferation, as well as an increased proliferation of the in situ ILC precursors to compensate their retention in the bone marrow.

S-5 : Critical mucosal protection by the complex innate lymphoid cell network

Innate lymphoid cells are an expanding family of lymphocytes that are prominently represented at mucosal surfaces and have been significantly implicated in the protection of epithelial barriers. In infection, ILCs contribute particularly to the early detection of pathogens and shape the subsequent adaptive immune response. Understanding the development of this innate population is necessary to provide insight to their functions during disease. We have investigated the network of transcription factors and found that Tbx21, Tcf7 and Nfil3 are all essential for the differentiation of ILC1, 2 and 3. While Tcf7 and Nfil3 were necessary for the generation of all ILC subsets, Tbx21 was crucial for the terminal differentiation and maturation of NCR + ILC3. These findings revealed key roles for specific transcription factors in individual cell types necessary for mucosal protection.

A committed hemopoietic precursor to innate lymphoid cells (HEM3P.277)

Abstract Innate lymphoid cells specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers. Their growing diversity and close similarities with previously characterized NK and LTi cells have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 based solely on cytokine properties. Elucidating their development pathways is essential to resolve their lineage relationships and dissect their respective functions in disease and homeostasis. Using lineage tracing and transfer studies, we identified and characterized a novel subset of lymphoid precursors in fetal liver and adult bone marrow that transiently expressed high amounts of PLZF, a transcription factor previously associated with NKT cell development. PLZF-high precursors were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 lineage differentiation potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1+ DX5- ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the frequency and function of several ILC subsets, but did not affect LTi or NK cells. These findings establish novel lineage relationships between ILC, NK and LTi cells and identify the elusive precursor to ILC, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.

Transcription factors controlling development and function of innate lymphoid cells

Innate lymphoid cells (ILCs) are a heterogeneous group of lymphocytes, which play an important role in tissue homeostasis at epithelial surfaces. They are scarce in spleen and lymph nodes, but substantial numbers can be found in the intestinal mucosa even at steady state. There, they represent the first line of defence against invading pathogens and contribute to lymphorganogenesis, tissue repair and, when inappropriately activated, immune pathology. Lineage-specific development, function and maintenance of these cells depend on a restricted set of transcription factors that partially emerged as a result of diversification and selection during vertebrate evolution. The differential expression of transcription factors regulates unique developmental programs, which endow the different ILC subsets with specific effector functions. Despite this division of labour, ILCs are considered to share a common origin, as they all are progeny of the common lymphoid progenitor, rely on the common γ-chain (γc) used by various cytokine receptors and show a developmental requirement for the transcriptional regulator Id2 (inhibitor of DNA binding 2). Here, we review the transcriptional programs required for the development and function of ILCs and give an overview of the evolution of transcription factors and cytokines expressed by ILCs.

Gata3 drives development of RORγt+ group 3 innate lymphoid cells

Group 3 innate lymphoid cells (ILC3) include IL-22-producing NKp46(+) cells and IL-17A/IL-22-producing CD4(+) lymphoid tissue inducerlike cells that express RORγt and are implicated in protective immunity at mucosal surfaces. Whereas the transcription factor Gata3 is essential for T cell and ILC2 development from hematopoietic stem cells (HSCs) and for IL-5 and IL-13 production by T cells and ILC2, the role for Gata3 in the generation or function of other ILC subsets is not known. We found that abundant GATA-3 protein is expressed in mucosa-associated ILC3 subsets with levels intermediate between mature B cells and ILC2. Chimeric mice generated with Gata3-deficient fetal liver hematopoietic precursors lack all intestinal RORγt(+) ILC3 subsets, and these mice show defective production of IL-22 early after infection with the intestinal pathogen Citrobacter rodentium, leading to impaired survival. Further analyses demonstrated that ILC3 development requires cell-intrinsic Gata3 expression in fetal liver hematopoietic precursors. Our results demonstrate that Gata3 plays a generalized role in ILC lineage determination and is critical for the development of gut RORγt(+) ILC3 subsets that maintain mucosal barrier homeostasis. These results further extend the paradigm of Gata3-dependent regulation of diversified innate ILC and adaptive T cell subsets.

Regulatory roles of dermal type 2 innate lymphoid cells

Regulatory roles of dermal type 2 innate lymphoid cells

OP0239 Phenotypic and Molecular Profile of Innate Lymphoid Cells in Chronic Synovial Inflammation

BackgroundInnate lymphoid cells (ILCs) represent a novel family of effector and regulatory cells in innate immunity and tissue remodelling [1]. The family comprises several phenotypically and functionally distinct subsets that...

Distinct requirements for T-bet in gut innate lymphoid cells

Interleukin (IL)-22-producing innate lymphoid cells (ILCs; ILC22) comprise a heterogeneous population of cells that are dependent on the transcription factor retinoid-related orphan γt (RORγt) and are critical for barrier function of the intestinal mucosa. A distinct ILC22 subset expresses the natural cytotoxicity receptor NKp46 (NKp46+ ILC22); however, the factors that contribute to the generation of this population versus other subsets are largely unknown. Herein, we show that T-bet (encoded by Tbx21) was highly expressed in NKp46+ ILC22, a feature shared by all NKp46+ cells present in the intestine but not by other IL-22-producing populations. Accordingly, the absence of T-bet resulted in loss of NKp46+ ILC22 in the intestinal lamina propria. The residual NKp46+ ILC22 present in Tbx21(-/-) mice showed a marked reduction of Rorγt expression and impairment in IL-22 production. Generation and functions of gut NK1.1+ cells were also altered. Bone marrow chimera experiments revealed a cell-intrinsic requirement for T-bet in these subsets and competitive reconstitution experiments revealed roles for T-bet in multiple ILC subsets. Thus, T-bet has a general importance for ILC in the gut and plays a selective and critical role in the generation of NKp46+ ILC22.

Polycystic Kidney Disease

Group 2 innate lymphoid cells (ILC2s) are a subset of ILCs that play a protective role in the response to helminth infection, but they also contribute to allergic lung inflammation. Here, we report that the deletion of the ETS1 transcription factor in lymphoid cells resulted in a loss of ILC2s in the bone marrow and lymph nodes and that ETS1 promotes the fitness of the common progenitor of all ILCs. ETS1-deficient ILC2 progenitors failed to up-regulate messenger RNA for the E protein transcription factor inhibitor ID2, a critical factor for ILCs, and these cells were unable to expand in cytokine-driven in vitro cultures. In vivo, ETS1 was required for the IL-33–induced accumulation of lung ILC2s and for the production of the T helper type 2 cytokines IL-5 and IL-13. IL-25 also failed to elicit an expansion of inflammatory ILC2s when these cells lacked ETS1. Our data reveal ETS1 as a critical regulator of ILC2 expansion and cytokine production and implicate ETS1 in the regulation of Id2 at the inception of ILC2 development.