Innate Lymphoid Cells Progenitor Research Articles

Determination of the Fate and Function of Innate Lymphoid Cells Following Adoptive Transfer of Innate Lymphoid Cell Precursors.

Innate lymphoid cells are a heterogeneous family of tissue-resident and circulating lymphocytes that play an important role in host immunity. Recent studies have profiled the developmental pathways of mature ILCs and have identified ILC progenitors in the bone marrow through the use of transcription factor reporter mice. Here we describe methodology to identify and isolate bone marrow CHILP and ILC2 progenitor (ILC2P) cells based on cell surface marker expression for adoptive transfer into lymphopenic mice to track the fate of developing ILCs.

Dissecting human ILC heterogeneity: more than just three subsets.

Innate lymphoid cells (ILCs) have been divided into three distinct groups based on functional capacities, cytokine profiles and transcription factor expression. Studies performed mainly in mice have demonstrated the importance of ILCs in chronic inflammation, infection, allergy and cancer. In this review, we discuss the heterogeneity of human ILC and focus primarily on the taxonomy of human ILC cell subsets and their phenotypical and functional diversity. We summarize recent findings concerning the diversity of ILCs between and within the major subsets [natural killer (NK), ILC1, intra-epithelial ILC1 (ieILC1), ILC2, ILC3, lymphoid tissues inducer (LTi) and ILC progenitor (ILCP)], as well as the abundance of each in human tissues. We also discuss the similarities observed between groups of cells in term of receptors expressed and cytokines produced, and how these relate to the pleiotropic properties of each subset.

Development and differentiation of early innate lymphoid progenitors.

Early innate lymphoid progenitors (EILPs) have recently been identified in mouse adult bone marrow as a multipotential progenitor population specified toward innate lymphoid cell (ILC) lineages, but their relationship with other described ILC progenitors is still unclear. In this study, we examine the progenitor-successor relationships between EILPs, all-lymphoid progenitors (ALPs), and ILC precursors (ILCps). Functional, bioinformatic, phenotypical, and genetic approaches collectively establish EILPs as an intermediate progenitor between ALPs and ILCps. Our work additionally provides new candidate regulators of ILC development and clearly defines the stage of requirement of transcription factors key for early ILC development.

GATA3 Regulates the Development and Functions of Innate Lymphoid Cell Subsets at Multiple Stages.

Innate lymphoid cells (ILCs) are regarded as the innate counterpart of effector CD4 T helper (Th) cells. Just as Th cells, ILCs are classified into distinct subsets based on their functions that are delivered mainly through effector cytokine production. Both ILCs and Th cells play critical roles in various protective immune responses and inflammatory diseases. Similar to Th cell differentiation, the development of ILC subsets depends on several master transcription factors, among which GATA3 is critical for the development and maintenance of type 2 ILCs (ILC2s). However, GATA3 is expressed by all ILC subsets and ILC progenitors, albeit at different levels. In a striking parallel with GATA3 function in T cell development and differentiation, GATA3 also has multiple functions in different ILCs at various stages. In this review, I will discuss how quantitative and dynamic expression of GATA3 regulates the development and functions of ILC subsets.

E proteins are important for restraining the ILC2 differentiation potential in the thymus

Abstract Innate lymphoid cells (ILCs) have gained recognition as important regulators in various immune responses. Current paradigm states that all ILCs originate from common lymphoid progenitors (CLP) in the bone marrow through an array of downstream ILC progenitors. Id2, a member of the Id family that inhibits E protein transcription factors, is expressed in these ILC progenitors and is indispensable for ILC differentiation. Interestingly, we found that down-regulating E protein function in the thymus by Id1 overexpression or deletion of two E protein genes resulted in massive expansion of ILC2s in the thymus as well as other organs where ILC2 normally reside. The phenotypic thymic ILC2s expressed ILC2 signature genes and down-regulated T lineage-specific genes. Moreover, Id1 transgenic mice exhibited spontaneous infiltration of eosinophils and heightened type 2 immune responses to the allergen, papain, in the lung. Furthermore, these mice had increased ability in the expulsion of helminth parasites, N. Brasiliensis. These results raise an interesting question as to whether the thymus is a natural site for ILC2 production and the phenotypes of the mutant mice simply highlight such capacity. Indeed, we found that wild type DN1 and DN3 thymocytes can differentiate into IL-5 and IL-13-producing ILC2 cells in vitro. Ablation of E proteins in DN3 cells accelerated the differentiation while gain of E protein function inhibited the process. Together, our data reveal a new ILC2 differentiation program occurring in the thymus and a critical role of E proteins in restraining such a differentiation potential while ensuring proper T cell production.

Deciphering the Innate Lymphoid Cell Transcriptional Program

Innate lymphoid cells (ILCs) are enriched at mucosal surfaces, where they provide immune surveillance. All ILC subsets develop from a common progenitor that gives rise to pre-committed progenitors for each of the ILC lineages. Currently, the temporal control of gene expression that guides the emergence ofthese progenitors is poorly understood. We used global transcriptional mapping to analyze gene expression in different ILC progenitors. We identified PD-1 to be specifically expressed in PLZF+ ILCp and revealed that the timing and order of expression of the transcription factors NFIL3, ID2, and TCF-1 was critical. Importantly, induction of ILC lineage commitment required only transient expression of NFIL3 prior to ID2 and TCF-1 expression. These findings highlight the importance of the temporal program that permits commitment of progenitors to the ILC lineage, and they expand our understanding of the core transcriptional program by identifying potential regulators of ILC development.

Single-cell RNA-seq identifies a PD-1hi ILC progenitor and defines its development pathway.

Innate lymphoid cells (ILCs) functionally resemble T lymphocytes in cytotoxicity and cytokine production but lack antigen-specific receptors, and they are important regulators of immune responses and tissue homeostasis. ILCs are generated from common lymphoid progenitors, which are subsequently committed to innate lymphoid lineages in the α-lymphoid progenitor, early innate lymphoid progenitor, common helper innate lymphoid progenitor and innate lymphoid cell progenitor compartments. ILCs consist of conventional natural killer cells and helper-like cells (ILC1, ILC2 and ILC3). Despite recent advances, the cellular heterogeneity, developmental trajectory and signalling dependence of ILC progenitors are not fully understood. Here, using single-cell RNA-sequencing (scRNA-seq) of mouse bone marrow progenitors, we reveal ILC precursor subsets, delineate distinct ILC development stages and pathways, and report that high expression of programmed death 1 (PD-1hi) marked a committed ILC progenitor that was essentially identical to an innate lymphoid cell progenitor. Our data defined PD-1hiIL-25Rhi as an early checkpoint in ILC2 development, which was abolished by deficiency in the zinc-finger protein Bcl11b but restored by IL-25R overexpression. Similar to T lymphocytes, PD-1 was upregulated on activated ILCs. Administration of a PD-1 antibody depleted PD-1hi ILCs and reduced cytokine levels in an influenza infection model in mice, and blocked papain-induced acute lung inflammation. These results provide a perspective for exploring PD-1 and its ligand (PD-L1) in immunotherapy, and allow effective manipulation of the immune system for disease prevention and therapy.

Down-regulation of E protein activity augments an ILC2 differentiation program in the thymus

Abstract Innate lymphoid cells (ILCs) have gained recognition as important regulators in various immune responses. How these cells are generated is just beginning to be understood. Current paradigm states that all three subsets of ILCs originate from common lymphoid progenitors (CLP) in the bone marrow through an array of downstream ILC progenitors. Id2, a member of the Id family that inhibits the function E protein transcription factors, is expressed in these ILC progenitors and is indispensable for ILC differentiation. Unexpectedly, we found that specifically down-regulating E protein function in the thymus by either Id1 expression or conditional deletion of two E protein genes resulted in massive expansion of ILC2s in the thymus as well as other organs where ILC2 normally reside. Consequently, the mutant mice exhibit augmented spontaneous infiltration of eosinophils and heightened responses to allergen, papain, in the lung. Furthermore, these mice also display increased ability in the expulsion of helminth parasites, N. Brasiliensis. These results raise an interesting question as to whether the thymus is a natural site for ILC2 production and the phenotypes of our mutant mice simply highlight such capacity. Indeed, lineage tracing experiments using lck-Cre/ROSA26-stop-tdTomato mice demonstrate that tdTomoto specifically marks a fraction of ILC2s in the thymus as well as in the lung, suggesting that thymus-derived ILC2s preferentially home to the lung. We also found that thymic progenitors were able to differentiate into ILC2 cells in vitro. Together, our data reveal a new ILC2 differentiation program present in the thymus, which enriches our knowledge about ILC2 ontogeny and has potential biological significance.

Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling.

T and innate lymphoid cells (ILCs) share some aspects of their developmental programs. However, although Notch signaling is strictly required for T cell development, it is dispensable for fetal ILC development. Constitutive activation of Notch signaling, at the common lymphoid progenitor stage, drives T cell development and abrogates ILC development by preventing Id2 expression. By combining single-cell transcriptomics and clonal culture strategies, we characterize two heterogeneous α4β7-expressing lymphoid progenitor compartments. αLP1 (Flt3(+)) still retains T cell potential and comprises the global ILC progenitor, while αLP2 (Flt3(-)) consists of ILC precursors that are primed toward the different ILC lineages. Only a subset of αLP2 precursors is sensitive to Notch signaling required for their proliferation. Our study identifies, in a refined manner, the diversity of transitional stages of ILC development, their transcriptional signatures, and their differential dependence on Notch signaling.

ID: 156: Transcriptional control of innate lymphoid cell fate decisions

Innate lymphoid cells (ILCs) are a recently discovered family of innate lymphocytes that are substantially represented at mucosal surfaces and have been implicated in the protection of epithelial barriers. Various types of ILCs can be discriminated based on the expression of distinct transcription factors controlling the expression of a distinct set of cytokine genes endowing the various ILC subsets with a specific range of effector functions. Currently, three groups of ILCs are being recognized. Group 1 ILCs (ILC1s) are a diverse group of ILCs comprised of natural killer (NK) cells and other, poorly defined subsets of ILCs. It is believed that the ILC1 fate decision is controlled by the T-box transcription factor T-bet endowing ILC1s with the capability to produce large amounts of IFN- γ . ILC2s express high levels of GATA-3, produce IL-5 and IL-13 and have been involved in immunity to helminth infections and in the pathogenesis of allergic diseases. Group 3 ILCs developmentally depend on the transcription factor ROR γ t and produce the cytokines IL-22, IL-17A and IL-17F. ILC3s are believed to be involved in the protection against intestinal bacterial infections and, if inappropriately stimulated, can be important drivers of inflammatory disorders. The transcriptional programs and effector cytokines of the various ILC subsets strikingly resemble those of the various T helper cell effector fates suggesting that such transcriptional circuitry already formed in the evolutionary older innate immune system. The various ILC subsets are developmentally related as all ILC lineages depend on the transcriptional regulator Id2 (inhibitor of DNA binding 2) that interfers with E2 protein-controlled gene expression. This raises the important issue if ILCs may derive from a common ILC progenitor (CILP). Identification of such a progenitor would allow to identify the molecular signals required for the specification of the various ILC lineages. I will discuss progress towards our understanding of the molecular programs regulating ILC fate decisions and our current models of transcriptional stability and plasticity of ILC fates. Finally, I will discuss an unprecedented role of ILC3s in the protection against mucosal virus infections. Research in my lab is supported by grants from the European Research Council (ERC) and Deutsche Forschungsgemeinschaft.

TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow.

The cellular and molecular events that drive early innate lympoid cell (ILC) development remain poorly understood. We show that transcription factor TCF-1 is required for the efficient generation of all known adult ILC subsets and their precursors. Using novel reporter mice, we identified a new subset of early ILC progenitors (EILP) that expressed high amounts of TCF-1. EILP lacked efficient T and B lymphocyte potential, but efficiently gave rise to NK cells and all known adult helper-ILC lineages, indicating that they are the earliest identified ILC-committed progenitors. Our results suggest that upregulation of TCF-1 expression denotes the earliest stage of ILC fate specification. The discovery of EILP provides a basis to decipher additional signals that specify the ILC fate.

Inflammatory group 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2 cells) are able to produce type 2 cytokines and to mediate type 2 immune protection and tissue homeostasis. ILC2 cells have often been considered to be a single set of cells that respond to IL-33 and/or IL-25. Recent evidence now indicates that ILC2 cells can be grouped into two distinct subsets: homeostatic or natural ILC2s (nILC2 cells); and inflammatory ILC2 cells (iILC2 cells). nILC2 cells reside in barrier tissues and primarily respond to IL-33. They play critical roles not only in immune protection but also in tissue repair and beige fat biogenesis. iILC2 cells are not present in peripheral tissues in the steady state but can be elicited at many sites by helminth infection or IL-25 treatment. IL-25-elicited ilLC2 cells act as transient ILC progenitors with multipotency. They can be mobilized by distinct types of infections to develop into nILC2-like or ILC3-like cells, functioning in corresponding immune responses. The demonstration of the existence of iILC2 cells adds to our understanding of the complexity of ILC2 biology and makes necessary an analysis of the relationship between nILC2 cells and iILC2 cells.

The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development

Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.

Innate lymphoid cell development requires TOX-dependent generation of a common ILC progenitor (HEM5P.221)

Abstract Subtypes of innate lymphoid cells (ILC), defined by effector function and transcription factor expression, have recently been identified in both mice and humans. ILC are critical in maintaining barrier homeostasis and orchestrating cross-talk between the innate and adaptive arms of the immune system. In the adult, ILC derive from common lymphoid progenitors in bone marrow by passing through multiple ILC-specific progenitor stages, although transcriptional regulation of the developmental pathways involved in this process remains poorly defined. Previously, we showed that the nuclear DNA-binding protein TOX is required for development of lymphoid tissue inducer cells, a type of ILC3 required for lymph node organogenesis, and conventional natural killer cells, a type of ILC1. Here, we demonstrate that the production of multiple ILC lineages requires TOX, as a result of TOX-dependent development of common ILC progenitors. In vitro and ex vivo studies indicate that this deficit is cell intrinsic. Furthermore, using low-input RNA sequencing, we obtained robust whole transcriptome data from wild-type and TOX-deficient ILC progenitors. Comparative analysis demonstrated failure to induce various aspects of the ILC gene program in the absence of TOX, implicating this nuclear factor as a key early determinant of ILC lineage specification.

NFIL3 orchestrates the emergence of common helper innate lymphoid cell precursors.

Innate lymphoid cells (ILCs) are a family of effectors that originate from a common innate lymphoid cell progenitor. However, the transcriptional program that sets the identity of the ILC lineage remains elusive. Here, we show that NFIL3 is a critical regulator of the common helper-like innate lymphoid cell progenitor (CHILP). Cell-intrinsic Nfil3 ablation led to variably impaired development of fetal and adult ILC subsets. Conditional gene targeting demonstrated that NFIL3 exerted its function prior to ILC subset commitment. Accordingly, NFIL3 ablation resulted in loss of ID2(+) CHILP and PLZF(+) ILC progenitors. Nfil3 expression in lymphoid progenitors was under the control of the mesenchyme-derived hematopoietin IL-7, and NFIL3 exerted its function via direct Id2 regulation in the CHILP. Moreover, ectopic Id2 expression in Nfil3-null precursors rescued defective ILC lineage development invivo. Our data establish NFIL3 as a key regulator of common helper-like ILC progenitors as they emerge during early lymphopoiesis.

Deciphering the transcriptional switches of innate lymphoid cell programming: the right factors at the right time.

Innate lymphoid cells (ILCs) are increasingly recognised as an innate immune counterpart of adaptive TH cells. In addition to their similar effector cytokine production, there is a strong parallel between the transcription factors that control the differentiation of TH1, TH2 and TH17 cells and ILC Groups 1, 2 and 3, respectively. Here, we review the transcriptional circuit that specifies the development of a common ILC progenitor and its subsequent programming into distinct ILC groups. Notch, GATA-3, Nfil3 and Id2 are identified as early factors that suppress B and T cell potentials and are turned on in favour of ILC commitment. Natural killer cells, which are the cytotoxic ILCs, develop along a pathway distinct from the rest of the helper-like ILCs that are derived from a common progenitor to all helper-like innate lymphoid cells (CHILPs). PLZF− CHILPs give rise to lymphoid tissue inducer cells while PLZF+ CHILPs have multi-lineage potential and could give rise to ILCs 1, 2 and 3. Such lineage specificity is dictated by the controlled expression of T-bet, RORα, RORγt and AHR. In addition to the type of transcription factors, the developmental stages at which these factors are expressed are crucial in specifying the fate of the ILCs.

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.

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity

The impact of the nutritional status during foetal life in the overall health of adults has been recognised1. However dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs (SLOs) occurs during embryogenesis and is considered to be developmentally programmed2,3. SLO formation dependents on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells2,3,4,5. Here we show that foetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero pre-setting the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi differentiation was controlled by maternal retinoid intake and foetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, while RA receptors directly regulated the Rorc locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

A committed precursor to innate lymphoid cells

Innate lymphoid cells (ILC) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers.1–9 Their diversity and similarities with previously characterized NK cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 based solely on cytokine properties,10 but their developmental pathways and lineage relationships remain elusive. 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.11,12 PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 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 development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of Id2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages.13 These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILC, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.