Innate lymphoid cells and cancer.

The innate lymphoid cell (ILC) family is composed of natural killer (NK) cells, ILC1, ILC2 and ILC3, which participate in immune responses to virus, bacteria, parasites and transformed cells. ILC1, ILC2 and ILC3 subsets are mostly tissue-resident, and are profoundly imprinted by their organ of residence. They exhibit pleiotropic effects, driving seemingly paradoxical responses such as tissue repair and, alternatively, immunopathology toward allergens and promotion of tumorigenesis. Despite this, a trickle of studies now suggests that non-NK ILCs may not be overwhelmingly tumorigenic and could potentially be harnessed to drive anti-tumor responses. Here, we examine the pleiotropic behavior of ILCs in cancer and begin to unravel the gap in our knowledge that exposes a new horizon for thinking about modifying ILCs and targeting them for immunotherapy.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Author Correction: The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

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

The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.

Complementarity and redundancy of IL-22-producing innate lymphoid cells.

Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.

CIS is a potent checkpoint in NK cell-mediated tumor immunity.

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15.

The inhibitor of DNA binding 2 (Id2) is essential for natural killer (NK) cell development with its canonical role being to antagonize E-protein function and alternate lineage fate. Here we have identified a key role for Id2 in regulating interleukin-15 (IL-15) receptor signaling and homeostasis of NK cells by repressing multiple E-protein target genes including Socs3. Id2 deletion in mature NK cells was incompatible with their homeostasis due to impaired IL-15 receptor signaling and metabolic function and this could be rescued by strong IL-15 receptor stimulation or genetic ablation of Socs3. During NK cell maturation, we observed an inverse correlation between E-protein target genes and Id2. These results shift the current paradigm on the role of ID2, indicating that it is required not only to antagonize E-proteins during NK cell commitment, but constantly required to titrate E-protein activity to regulate NK cell fitness and responsiveness to IL-15.

Natural Killer Cells and Type 1 Innate Lymphoid Cells in Hepatocellular Carcinoma: Current Knowledge and Future Perspectives.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) are specific innate lymphoid cell subsets that are key for the detection and elimination of pathogens and cancer cells. In liver, while they share a number of characteristics, they differ in many features. These include their developmental pathways, tissue distribution, phenotype and functions. NK cells and ILC1 contribute to organ homeostasis through the production of key cytokines and chemokines and the elimination of potential harmful bacteria and viruses. In addition, they are equipped with a wide range of receptors, allowing them to detect "stressed cells' such as cancer cells. Our understanding of the role of innate lymphoid cells in hepatocellular carcinoma (HCC) is growing owing to the development of mouse models, the progress in immunotherapeutic treatment and the recent use of scRNA sequencing analyses. In this review, we summarize the current understanding of NK cells and ILC1 in hepatocellular carcinoma and discuss future strategies to take advantage of these innate immune cells in anti-tumor immunity. Immunotherapies hold great promise in HCC, and a better understanding of the role and function of NK cells and ILC1 in liver cancer could pave the way for new NK cell and/or ILC1-targeted treatment.

Constitutive overexpression of TNF in BPSM1 mice causes iBALT and bone marrow nodular lymphocytic hyperplasia.

BPSM1 (Bone phenotype spontaneous mutant 1) mice develop severe polyarthritis and heart valve disease as a result of a spontaneous mutation in the Tnf gene. In these mice, the insertion of a retrotransposon in the 3' untranslated region of Tnf causes a large increase in the expression of the cytokine. We have found that these mice also develop inducible bronchus-associated lymphoid tissue (iBALT), as well as nodular lymphoid hyperplasia (NLH) in the bone marrow. Loss of TNFR1 prevents the development of both types of follicles, but deficiency of TNFR1 in the hematopoietic compartment only prevents the iBALT and not the NLH phenotype. We show that the development of arthritis and heart valve disease does not depend on the presence of the tertiary lymphoid tissues. Interestingly, while loss of IL-17 or IL-23 limits iBALT and NLH development to some extent, it has no effect on polyarthritis or heart valve disease in BPSM1 mice.

NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology.

OBJECTIVES: NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans. METHODS: Here, we characterised a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model. RESULTS: The patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a preclinical model, where Nfil3-deficient mice upregulated IL-1ß production, with more severe arthritis symptoms on disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1ß as a pivotal cytokine. CONCLUSIONS: NFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1ß over-production.

Development, Homeostasis, and Heterogeneity of NK Cells and ILC1.

Natural killer (NK) cells are a population of cytotoxic innate lymphocytes that evolved prior to their adaptive counterparts and constitute one of the first lines of defense against infected or mutated cells. NK cells are rapidly activated, expressing an array of germ-line encoded receptors that allow them to scan for protein irregularities on cells and kill those deemed "altered-self." NK cells rapidly produce a broad range of cytokines and chemokines following activation by virus, bacterial, or parasitic infection and are thus key in orchestrating inflammation. NK cells have previously been viewed to represent a relatively homogeneous group of IFN-γ-producing cells that express the surface markers NK1.1 and natural killer cell p46-related protein (NKp46 or NCR1 encoded by Ncr1) and depend on the transcription factor T-bet for their development. Recently, a second subset of T-bet-dependent innate cells, the group 1 innate lymphoid cells (ILC1), has been discovered which share many attributes of conventional NK (cNK) cells. Despite the similarities between ILC1 and cNK cells , they differ in several important aspects including their localization, transcriptional regulation, and phenotype suggesting each subset has distinct origins and functions in immune responses. Previously, the ability to detect and spontaneously kill cells that exhibit "altered-self" which is central to tumor and viral immunity has been thought to be an attribute restricted solely to cNK cells. The identification of ILC1 challenges this notion and suggests that key contributions from ILC1 may have gone unrecognized. Thus, understanding the different rules that govern the behavior of ILC1 and cNK cells in immune responses may potentially open unexpected doorways to uncover novel strategies to manipulate these cells in treating disease. Here, we review recent advances in our understanding of peripheral cNK cell and ILC1 heterogeneity in terms of their development, phenotype, homeostasis, and effector functions.

Differentiation and diversity of subsets in group 1 innate lymphoid cells.

NK cells were first identified in 1975 and represent the prototypical group 1 innate lymphoid cell (ILC). More recently, the discovery of new members of the ILC family has highlighted the complexity of this innate lymphoid lineage. Importantly, it has been recognized that different subsets exist within the group 1 ILC, which have potential roles in mediating immune protection and immunosurveillance, and in regulating tissue homeostasis and inflammation. Here, we review the developmental relationships between the different group 1 ILC, which have been identified to date and discuss how heterogeneity within this expanding family may have arisen.

Id2 represses E2A-mediated activation of IL-10 expression in T cells.

Interleukin-10 (IL-10) is a key immunoregulatory cytokine that functions to prevent inflammatory and autoimmune diseases. Despite the critical role for IL-10 produced by effector CD8(+) T cells during pathogen infection and autoimmunity, the mechanisms regulating its production are poorly understood. We show that loss of the inhibitor of DNA binding 2 (Id2) in T cells resulted in aberrant IL-10 expression in vitro and in vivo during influenza virus infection and in a model of acute graft-versus-host disease (GVHD). Furthermore, IL-10 overproduction substantially reduced the immunopathology associated with GVHD. We demonstrate that Id2 acts to repress the E2A-mediated trans-activation of the Il10 locus. Collectively, our findings uncover a key regulatory role of Id2 during effector T cell differentiation necessary to limit IL-10 production by activated T cells and minimize their suppressive activity during the effector phase of disease control.

X-Chromosome complement and estrogen receptor signaling independently contribute to the enhanced TLR7-mediated IFN-α production of plasmacytoid dendritic cells from women.

Human plasmacytoid dendritic cells (pDCs) play a major role in innate immunity through the production of type I IFNs after TLR engagement by pathogens. Sex-based differences in the innate function of human pDCs have been established, with pDCs from women exhibiting enhanced TLR7-mediated IFN-α production as compared with pDCs from males. In mice, we recently provided evidence for a role of estrogens as a positive regulator of pDC innate functions through cell-intrinsic estrogen receptor α signaling, but did not exclude a role for other X-linked factors, particularly in human pDCs. In this study, we investigated the respective contribution of X chromosome dosage and sex hormones using a humanized mouse model in which male or female NOD-SCID-ß2m(-/-) were transplanted with human progenitor cells purified from either male or female cord blood cells. We showed that, in response to TLR7 ligands, the frequency of IFN-α- and TNF-α-producing pDCs from either sex was greater in female than in male host mice, suggesting a positive role for estrogens. Indeed, blockade of estrogen receptor signaling during pDC development in vitro inhibited TLR7-mediated IFN-α production by human pDCs, which expressed both ESR1 and ESR2 genes. Interestingly, we also found that X chromosome dosage contributed to this sex bias as female pDCs have an enhanced TLR7-mediated IFN-α response as compared with male ones, irrespective of the sex of the recipient mice. Together, these results indicate that female sex hormones, estrogens, and X chromosome complement independently contribute to the enhanced TLR7-mediated IFN-α response of pDCs in women.

Differential requirement for Nfil3 during NK cell development.

NK cells can be grouped into distinct subsets that are localized to different organs and exhibit a different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor Nfil3 (E4bp4) is essential for bone marrow-derived NK cell development, but it is not clear whether Nfil3 is equally important for all NK cell subsets or how it induces NK lineage commitment. In this article, we show that Nfil3 is required for the formation of Eomes-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL(+) Eomes(-) NK cells develop independently of Nfil3. Loss of Nfil3 during the development of bone marrow-derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3(-/-) progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cells by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3.

CD8α+ DCs can be induced in the absence of transcription factors Id2, Nfil3, and Batf3.

Antiviral immunity and cross-presentation is mediated constitutively through CD8α+ and CD103+ DCs. Development of these DC subsets is thought to require the transcription factors Irf8, Id2, Nfil3, and Batf3, although how this network is regulated is poorly defined. We addressed the nature of the differentiation blocks observed in the absence of these factors and found that although all 4 factors are required for CD103+ DC development, only Irf8 is essential for CD8α+ DCs. CD8α+ DCs emerged in the absence of Id2, Nfil3 and Batf3 in short-term bone marrow reconstitution. These "induced" CD8α+ DCs exhibit several hallmarks of classic CD8α+ DCs including the expression of CD24, Tlr3, Xcr1, Clec9A, and the capacity to cross-present soluble, cell-associated antigens and viral antigens even in the absence of Batf3. Collectively, these results uncover a previously undescribed pathway by which CD8α+ DCs emerge independent of Id2, Nfil3, and Batf3, but dependent on Irf8.

TCF-1 controls ILC2 and NKp46+RORγt+ innate lymphocyte differentiation and protection in intestinal inflammation.

Innate lymphocyte populations play a central role in conferring protective immunity at the mucosal frontier. In this study, we demonstrate that T cell factor 1 (TCF-1; encoded by Tcf7), a transcription factor also important for NK and T cell differentiation, is expressed by multiple innate lymphoid cell (ILC) subsets, including GATA3(+) nuocytes (ILC2) and NKp46(+) ILCs (ILC3), which confer protection against lung and intestinal inflammation. TCF-1 was intrinsically required for the differentiation of both ILC2 and NKp46(+) ILC3. Loss of TCF-1 expression impaired the capacity of these ILC subsets to produce IL-5, IL-13, and IL-22 and resulted in crippled responses to intestinal infection with Citrobacter rodentium. Furthermore, a reduction in T-bet expression required for Notch-2-dependent development of NKp46(+) ILC3 showed a dose-dependent reduction in TCF-1 expression. Collectively, our findings demonstrate an essential requirement for TCF-1 in ILC2 differentiation and reveal a link among Tcf7, Notch, and Tbx21 in NKp46(+) ILC3 development.

Estradiol promotes functional responses in inflammatory and steady-state dendritic cells through differential requirement for activation function-1 of estrogen receptor α.

17ß-Estradiol (E2) has been shown to regulate GM-CSF- or Flt3 ligand-driven dendritic cell (DC) development through estrogen receptor (ER) α signaling in myeloid progenitors. ERα regulates transcription of target genes through two distinct activation functions (AFs), AF-1 and AF-2, whose respective involvement varies in a cell type- or tissue-specific manner. In this study, we investigated the role of ERα AFs in the development and effector functions of inflammatory DCs, steady-state conventional DCs, and plasmacytoid DCs (pDC), using mouse lacking either AF-1 or AF-2. In agreement with previous works, we showed that E2 fostered the differentiation and effector functions of inflammatory DCs through ERα-dependent upregulation of IFN regulatory factor (IRF)-4 in GM-CSF-stimulated myeloid progenitors. Interestingly, whereas AF-1 was required for early IRF-4 upregulation in DC precursors, it was dispensable to enhance IRF-4 expression in differentiated DCs to a level compatible with the development of the more functional Ly6C(-) CD11b(+) DC subset. Presence of E2 had no effect on progenitors from either knock-in mice with 7-aa deletion in helix 12 of ERα, lacking AF-2, or ERα(-/-) mice. By contrast, in Flt3 ligand-driven DC differentiation, activation of AF-1 domain was required to promote the development of more functionally competent conventional DCs and pDCs. Moreover, lack of ERα AF-1 blunted the TLR7-mediated IFN-α response of female pDCs in vivo. Thus, our study demonstrates that ERα uses AF-1 differently in steady-state and inflammatory DC lineages to regulate their innate functions, suggesting that selective ER modulators could be used to target specific DC subsets.

The TLR-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor α signaling.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type I interferons (IFN-α/ß) in response to viral or endogenous nucleic acids through activation of their endosomal Toll-like receptors (TLR-7 and TLR-9). Enhanced TLR-7-mediated IFN-α production by pDCs in women, compared with men, has been reported, but whether sex hormones, such as estrogens, are involved in this sex-based difference is unknown. Here we show, in humanized mice, that the TLR-7-mediated response of human pDCs is increased in female host mice relative to male. In a clinical trial, we establish that treatment of postmenopausal women with 17ß-estradiol markedly enhances TLR-7- and TLR-9-dependent production of IFN-α by pDCs stimulated by synthetic ligands or by nucleic acid-containing immune complexes. In mice, we found exogenous and endogenous estrogens to promote the TLR-mediated cytokine secretion by pDCs through hematopoietic expression of estrogen receptor (ER) α. Genetic ablation of ERα gene in the DC lineage abrogated the enhancing effect of 17ß-estradiol on their TLR-mediated production of IFN-α, showing that estrogens directly target pDCs in vivo. Our results uncover a previously unappreciated role for estrogens in regulating the innate functions of pDCs, which may account for sex-based differences in autoimmune and infectious diseases.