Innate lymphoid cell development.

Innate lymphoid cells (ILCs) mainly reside at barrier surfaces and regulate tissue homeostasis and immunity. ILCs are divided into 3 groups, group 1 ILCs, group 2 ILCs, and group 3 ILC3, on the basis of their similar effector programs to T cells. The development of ILCs from lymphoid progenitors in adult mouse bone marrow has been studied in detail, and multiple ILC progenitors have been characterized. ILCs are mostly tissue-resident cells that develop in the perinatal period. More recently, ILC progenitors have also been identified in peripheral tissues. In this review, we discuss the stepwise transcription factor-directed differentiation of mouse ILC progenitors into mature ILCs, the critical time windows in ILC development, and the contribution of bone marrow versus tissue ILC progenitors to the pool of mature ILCs in tissues.

Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets.

Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor-related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2- population different from conventional IL-18Rα-ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

Identification of Group 2 Innate Lymphoid Cells in Mouse Lung, Liver, Small Intestine, Bone Marrow, and Mediastinal and Mesenteric Lymph Nodes.

Innate lymphoid cells (ILCs) are a heterogeneous family of lymphocytes that populate barrier and non-barrier tissues. ILCs regulate immune responses to pathogens and commensals but also sustain metabolic homeostasis, tissue remodeling after injury and establish dialogue with the nervous system. ILCs rapidly become activated in the absence of adaptive antigen receptors by responding to signaling molecules provided by hematopoietic or non-hematopoietic cells. Here we provide protocols designed for processing the lung, liver, small intestine, bone marrow, mediastinal and mesenteric lymph nodes in order to obtain a purified leukocyte fraction of cells, in which ILC2 enrichment is optimized. In addition, we describe in detail the methodologies used to activate ILC2s and the assays necessary for the detection of their effector cytokines. We highlight the differences in ILC2 characterization within distinct tissues that we have recently identified. © 2019 by John Wiley & Sons, Inc.

Type 2 Innate Lymphocytes Actuate Immunity Against Tumours and Limit Cancer Metastasis.

Type 2 innate lymphoid cells (ILC2) potentiate immune responses, however, their role in mediating adaptive immunity in cancer has not been assessed. Here, we report that mice genetically lacking ILC2s have significantly increased tumour growth rates and conspicuously higher frequency of circulating tumour cells (CTCs) and resulting metastasis to distal organs. Our data support the model that IL-33 dependent tumour-infiltrating ILC2s are mobilized from the lungs and other tissues through chemoattraction to enter tumours, and subsequently mediate tumour immune-surveillance by cooperating with dendritic cells to promote adaptive cytolytic T cell responses. We conclude that ILC2s play a fundamental, yet hitherto undescribed role in enhancing anti-cancer immunity and controlling tumour metastasis.

Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity by targeting tight junctions through IL-13 in asthmatic patients.

BACKGROUND: Bronchial epithelial barrier leakiness and type 2 innate lymphoid cells (ILC2s) have been separately linked to asthma pathogenesis; however, the influence of ILC2s on the bronchial epithelial barrier has not been investigated previously. OBJECTIVE: We investigated the role of ILC2s in the regulation of bronchial epithelial tight junctions (TJs) and barrier function both in bronchial epithelial cells of asthmatic patients and healthy subjects and general innate lymphoid cell- and ILC2-deficient mice. METHODS: Cocultures of human ILC2s and bronchial epithelial cells were used to determine transepithelial electrical resistance, paracellular flux, and TJ mRNA and protein expressions. The effect of ILC2s on TJs was examined by using a murine model of IL-33-induced airway inflammation in wild-type, recombination-activating gene 2 (Rag2)-/-, Rag2-/-Il2rg-/-, and Rorasg/sg mice undergoing bone marrow transplantation to analyze the in vivo relevance of barrier disruption by ILC2s. RESULTS: ILC2s significantly impaired the epithelial barrier, as demonstrated by reduced transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran permeability in air-liquid interface cultures of human bronchial epithelial cells. This was in parallel to decreased mRNAs and disrupted protein expression of TJ proteins and was restored by neutralization of IL-13. Intranasal administration of recombinant IL-33 to wild-type and Rag2-/- mice lacking T and B cells triggered TJ disruption, whereas Rag2-/-Il2rg-/- and Rorasg/sg mice undergoing bone marrow transplantation that lack ILC2s did not show any barrier leakiness. Direct nasal administration of IL-13 was sufficient to induce deficiency in the TJ barrier in the bronchial epithelium of mice in vivo. CONCLUSION: These data highlight an essential mechanism in asthma pathogenesis by demonstrating that ILC2s are responsible for bronchial epithelial TJ barrier leakiness through IL-13.

Immunological Memory of Group 2 Innate Lymphoid Cells.

Immunological memory has long been described as a property of the adaptive immune system that results in potent responses on exposure to an antigen encountered previously. While this definition appears to exclude cells that do not express antigen receptors, recent studies have shown that innate immune cells, including natural killer (NK) cells, macrophages, and, more recently, group 2 innate lymphoid cells (ILC2s) can record previous activations and respond more vigorously on reactivation. Here we review the similarities and differences between these forms of memory and the underlying mechanisms. Based on these insights, we propose to revise the definition of immunological memory, as the capacity to remember being previously activated and respond more efficiently on reactivation regardless of antigen specificity.

G9a regulates group 2 innate lymphoid cell development by repressing the group 3 innate lymphoid cell program.

Innate lymphoid cells (ILCs) are emerging as important regulators of homeostatic and disease-associated immune processes. Despite recent advances in defining the molecular pathways that control development and function of ILCs, the epigenetic mechanisms that regulate ILC biology are unknown. Here, we identify a role for the lysine methyltransferase G9a in regulating ILC2 development and function. Mice with a hematopoietic cell-specific deletion of G9a (Vav.G9a(-/-) mice) have a severe reduction in ILC2s in peripheral sites, associated with impaired development of immature ILC2s in the bone marrow. Accordingly, Vav.G9a(-/-) mice are resistant to the development of allergic lung inflammation. G9a-dependent dimethylation of histone 3 lysine 9 (H3K9me2) is a repressive histone mark that is associated with gene silencing. Genome-wide expression analysis demonstrated that the absence of G9a led to increased expression of ILC3-associated genes in developing ILC2 populations. Further, we found high levels of G9a-dependent H3K9me2 at ILC3-specific genetic loci, demonstrating that G9a-mediated repression of ILC3-associated genes is critical for the optimal development of ILC2s. Together, these results provide the first identification of an epigenetic regulatory mechanism in ILC development and function.

Common-Lymphoid-Progenitor-Independent Pathways of Innate and T Lymphocyte Development.

All lymphocytes are thought to develop from common lymphoid progenitors (CLPs). However, lymphoid-primed multipotent progenitors (LMPPs) are more efficient than CLPs in differentiating into T cells and group 2 innate lymphoid cells (ILC2s). Here, we have divided LMPPs into CD127(-) (LMPP-s) and CD127(+) (LMPP+s) subsets and compared them with Ly6D(-) and Ly6D(+) CLPs. Adult LMPP+s differentiated into T cells and ILCs more rapidly and efficiently than other progenitors in transplantation assays. The development of T cells and ILC2s is highly active in the neonatal period. Neonatal CLPs are rare and, unlike prominent neonatal LMPP+s, do not efficiently differentiate into T cells and ILC2s. ILC2s generated in the neonatal period are long lived and persist in adult tissues. These results suggest that some ILCs and T cells may develop from LMPP+s via CLP-independent pathways.

UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.

Lung ILC2s link innate and adaptive responses in allergic inflammation.

How allergens trigger the T helper 2 (Th2) response that characterizes allergic lung inflammation is not well understood. Epithelium-derived alarmins released after an allergen encounter activate the innate immune system, including group 2 innate lymphoid cells (ILC2s) which produce the type 2 interleukins IL-5 and IL-13. It has been recently shown that ILC2-derived cytokines are responsible not only for the innate responses underlying allergic inflammation but also for the initiation of the adaptive Th2 response. We review the role of lung ILC2s in the development of allergic inflammation and, in the context of recent findings, propose a common pathway wherein ILC2s, activated by the epithelium-derived cytokine IL-33, link the innate and the adaptive responses after allergen encounter in the lung.

Group 2 innate lymphoid cells are critical for the initiation of adaptive T helper 2 cell-mediated allergic lung inflammation.

Naive CD4(+) T cell differentiation into distinct subsets of T helper (Th) cells is a pivotal process in the initiation of the adaptive immune response. Allergens predominantly stimulate Th2 cells, causing allergic inflammation. However, why allergens induce Th2 cell differentiation is not well understood. Here we show that group 2 innate lymphoid cells (ILC2s) are required to mount a robust Th2 cell response to the protease-allergen papain. Intranasal administration of papain stimulated ILC2s and Th2 cells, causing allergic lung inflammation and elevated immunoglobulin E titers. This process was severely impaired in ILC2-deficient mice. Whereas interleukin-4 (IL-4) was dispensable for papain-induced Th2 cell differentiation, ILC2-derived IL-13 was critical as it promoted migration of activated lung dendritic cells into the draining lymph node where they primed naive T cells to differentiate into Th2 cells. Papain-induced ILC2 activation and Th2 cell differentiation was IL-33-dependent, suggesting a common pathway in the initiation of Th2 cell responses to allergen.

Retinoic-acid-receptor-related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation.

Natural helper (NH) cells are innate lymphoid cells (ILCs) that produce T helper-2 (Th2)-cell-type cytokines in the lung- and gut-associated lymphoid tissues. Currently, the lineage relationship between NH cells in different tissues and between NH cells and interleukin-22 (IL-22)-producing retinoic-acid-receptor-related orphan receptor (ROR)γt-positive ILCs is unclear. Here, we report that NH cells express RORα, but not RORγt. RORα-deficient, but not RORγt-deficient, mice lacked NH cells in all tissues, whereas all other lymphocytes, including RORγt(+) ILCs, were unaffected. NH-cell-deficient mice generated by RORα-deficient bone-marrow transplantation had normal Th2 cell responses but failed to develop acute lung inflammation in response to protease allergen, thus confirming the essential role of NH cells in allergic lung inflammation. We have also identified RORα-dependent NH cell progenitors in the bone marrow. Thus, all NH cells belong to a unique RORα-dependent cell lineage separate from other lymphoid cell lineages.

Lymphoid progenitors in normal mouse lymph nodes develop into NK cells and T cells in vitro and in vivo.

We have identified a population of normal mouse LN cells, termed LN lymphoid progenitor (LNLP), resembling common lymphoid progenitor (CLP) in the bone marrow. LNLPs lack lineage markers and express CD127, low levels of CD117 (c-Kit), and Sca-1, but lack fms-related tyrosine kinase 3. They efficiently differentiate in vitro into natural killer (NK) cells and T cells, but not mature B cells. LNLPs injected into nonirradiated lymphopenic mice that have no LN develop into mostly splenic T cells with low numbers of NK cells and B cells. When injected into irradiated mice, they generate NK cells and T cells, but not B cells, in the LN. By contrast, bone marrow CLPs develop into mostly B cells with very small numbers of T and NK cells in recipients' spleen and LN. LNLPs have NK and T-cell potentials, but little B-cell potential, and they can develop into NK cells within the LN of normal mice, but their contribution to the T-cell lineage is unknown.

LAK cell therapy of AML: not to be lost in translation.

Expanding highly cytotoxic lymphokine activated natural killer cells and cytokine induced killer T cells by Good Manufacturing Practice-quality cultures is an important step toward effective use of those killer cells to treat acute myeloid leukemia patients receiving stem cell transplantation. Elucidating how those killer cells recognize leukemia stem cells will be the next challenge.

B-cell co-receptor CD72 is expressed on NK cells and inhibits IFN-gamma production but not cytotoxicity.

NK cells have two main functions, namely cell-mediated cytotoxicity and production of cytokines. Multiple inhibitory receptors that regulate NK-cell cytotoxicity have been characterized whereas little is known about receptors regulating cytokine production. Here we report that CD72, which is considered to be an important co-receptor regulating B-cell activation, is also expressed on mouse NK cells. NK cells expressing high levels of CD72, upon stimulation with IL-12 and IL-18 or target cells, produce significantly less IFN-gamma than those expressing low levels of CD72, whereas both subsets are equally cytotoxic. Ectopic expression of CD72 in the murine NK-cell line KY2 inhibits cytokine-induced IFN-gamma production, and the inhibitory effect is diminished by mutations in the inhibitory motifs in the intracellular domain or replacement of the extracellular domain of CD72. Thus, CD72 is an inhibitory receptor on NK cells regulating cytokine production.

A dual role for talin in NK cell cytotoxicity: activation of LFA-1-mediated cell adhesion and polarization of NK cells.

LFA-1 is critical for NK cell cytotoxicity because it mediates adhesion of NK cells to target cells. Talin is thought to associate with the cytoplasmic tail of LFA-1 and activates its ligand-binding function. In this study, we report that talin is also required for LFA-1-mediated outside-in signaling leading to NK cell polarization. NK cells generated from talin1-deficient murine embryonic stem cells are defective in LFA-1-mediated adhesion. Although exogenously added manganese activates LFA-1 on talin-deficient NK cells and induces conjugate formation with target cells, their LFA-1-dependent cytotoxicity is impaired. Binding of ICAM-1-coated beads to wild-type NK cells induces reorganization of the actin cytoskeleton and coligation of the activating receptor NKG2D induces polarization of cytotoxic granules, whereas talin1-deficient NK cells fail to polarize with or without NKG2D coligation. Thus, talin1 plays a dual role in NK cell cytotoxicity, first by activation of LFA-1-mediated adhesion and then via LFA-1-induced NK cell polarization.

Activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage.

Activation of calpains by calcium flux leading to talin cleavage is thought to be an important process of LFA-1 activation by inside-out signalling. Here, we tested the effects of the calcium ionophore ionomycin and calpain inhibitor calpeptin on LFA-1-mediated adhesion of a T cell hybridoma line, cytotoxic T cells and primary resting T cells. Ionomycin activated LFA-1-mediated adhesion of all three types of T cells, and calpeptin inhibited the effects of ionomycin. However, calpeptin also inhibited activation of LFA-1 by PMA, which did not induce calcium flux. Cleavage of talin was undetectable in ionomycin-treated T cells. Furthermore, treatment with ionomycin and calpeptin induced apoptosis of T cells. Inhibitors of phosphatidyl Inositol-3 kinase inhibited activation of LFA-1 by ionomycin, but not by PMA, whereas the protein kinase C inhibitor inhibited the effects of PMA, but not ionomycin. Thus, activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage.

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro.

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.

A role for DNA hypomethylation and histone acetylation in maintaining allele-specific expression of mouse NKG2A in developing and mature NK cells.

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However, the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study, we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow, in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells, NK progenitors, and NKG2A-negative NK cells), and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore, we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally, we show that acetylated histones are associated with the CpG-rich region in NKG2A positive, but not negative, cell lines, and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription, but cotreatment with both drugs resulted in a significantly greater induction, suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells.

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells.

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.