The adaptive immune system has evolved antigen receptor diversity to cope with a large variety of pathogens and non-self antigens. It is well-established that, depending on the nature of the invader and the signals coming from the surrounding environment, the adaptive immune system mounts a specific effector T helper (Th) response that serves to control the trespasser and re-establish homeostasis. Functionally, Th1 eliminate intracellular pathogens, Th2 are critical in promoting the eradication of helminthes, and Th17 control fungal and bacterial infections (1).

In the last few years, it has become evident that the innate immune system displays a similar strategy in order to ensure a first line of defense against intruders via innate lymphoid cells (ILCs). Broadly, ILCs are defined by their lymphoid lineage and their lack of RAG-mediated recombined antigen receptors. Mirroring the Th subsets, ILC populations diverge from one another in their reliance on unique transcription factors and production of signature cytokines. In general, ILCs provide primary border patrol at mucosal surfaces sensing changes in the microenvironment and rapidly responding to insults by producing specific cytokines that limit the damage caused by the attacking pathogen or favor its clearance and elimination.

Until ILCs came into the spotlight, conventional NK cells (cNKs) (i.e., blood circulating CD56⁺ cells in human and splenic, lymph node, and bone marrow NKp46⁺NK1.1⁺ cells in mouse) were the only innate immune cells known to respond to cytokines produced by antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages (2–4).

Natural killer (NK) cells can rapidly release IFN-γ in response to IL-12 and IL-18 and/or type 1 IFN, which are produced when viruses and bacteria infect or come into contact with APCs. Because of their capacity to immediately respond to cytokines, cNK cells can be viewed as the prototype of all ILC subsets. However, they have additional properties, such as cytotoxic ability, that set them apart from other ILCs and allow them to eliminate virally infected or tumor-transformed cells.

Here, we will review the lineage relationship between ILC subsets and cNKs based on key transcription factors that regulate their respective development as well as the current understanding their functional roles.

Innate lymphoid cells, like Th cells, are heterogeneous and currently grouped in three major subsets: ILC1, ILC2, and ILC3 (5).

Until recently, the general consensus was that the very same transcription factors that drive Th1, Th2, and Th17 commitment and differentiation also regulate ILC1, ILC2, and ILC3 development, respectively. Moreover, it seemed that cNKs and the other ILC groups significantly differed in their developmental requirement for transcription factors.

This concept has been recently challenged by a number of studies suggesting that developmental requirements for NK cell subsets and ILCs are more complex than originally anticipated (Figure 1). Here, we will highlight the most recent progresses in the field.

All ILC groups, including cNKs, depend on Id2 (47, 93). Id2 is a transcriptional repressor that, by blocking members of the E2 family of transcription factors, suppresses the B cell fate potential in a progenitor cells that develop downstream of the common lymphoid precursor (CLP). A recent study that took advantage of a reporter mouse expressing GFP under the control of the Id2 promoter to track Id2-expressing cells has shown that Id2 is not expressed in CLPs. Id2 is, however, expressed at very high level in all ILCs, including cNKs (94). In NK cells, Id2 is not required for the NK cell lineage specification, since development of CD122⁺NK1.1⁻ NK cell progenitors is not impaired in Id2-deficient mice. Nevertheless, Id2 expression in NK cells is required for acquisition of a fully mature phenotype. The few NK cells that develop in the absence of Id2 have a phenotype that closely resemble thymic NK cells, express high levels of CD127 and produce large amounts of IFN-γ (95). In committed B cells, the transcription factor EBF1 mediates Id2 suppression (96). According to the idea that Id2 is globally required for development of all ILCs, EBF1-deficient pro-B cells acquire the capacity to differentiate into T cells and into functionally competent ILC2s and ILC3s (97). Importantly, the generation of an Id2-reporter mouse has allowed the identification of a common ILC progenitor, or CHILP (common “helper-like” ILC lineages progenitor) (10) that can generate CD127⁺NKp46⁺ siLP ILC1, ILC2, and ILC3 in vivo and in vitro. This Lin⁻Id2⁺ progenitor also expresses CD127 and the integrin α4β7 but does not express CD25 (which is present on differentiated ILC2) or CD122, a marker of NK cell precursors. Intriguingly, this precursor is only slightly affected by IL-7 deficiency and can generate liver-resident CD49a⁺Eomes⁻ NK cells, but not cNKs (10). Further confirming the existence of a common ILC progenitor, an other recent study performing fate-mapping experiments shows that ILCs, including intraepithelial ILC1, ILC2, and NCR⁺ ILC3, derive from a common committed precursor present in fetal liver and adult bone marrow that transiently expresses the transcription factor PLZF (encoded by Zbtb16) (98). This transcription factor was known to be required for NKT development, but it is dispensable for cNK cells and CD4⁺ LTi-like cells (99). Reconstitution experiments with Zbtb16-deficient or -sufficient bone marrow cells in lethally irradiated mice indicated that Zbtb16 is absolutely required for ILC2s development only. On the contrary, it seems to be dispensable for NCR⁺ ILC3s reconstitution, despite its high expression in this subset. As Zbtb16 is controlled by Id2 (100), the PLZF-expressing common ILC precursor may be downstream of the Id2⁺CD127⁺CD25⁻α4β7⁺ CHILP.

Most likely downstream of the CHILP, all ILC3 subsets require the Th17 master regulator RORγt (101, 102). In the absence of RORγt, Lti, and Lti-like cells fail to develop and RORγt-deficient mice do not possess lymph nodes, PP, cryptopaches (CP) nor ILFs (103, 104), which derive from CP under signals coming from the microbiota through the nod-like receptor (NLR) NOD1 (105). ILC3s also require aryl hydrocarbon receptor (AHR) (106–108), which drives IL-22 secretion in Th17 cells (109, 110). In the absence of AHR, both Lti-like ILC3s and NCR⁺ ILC3s are severely diminished. In addition, small intestine CP and ILFs are absent, while colonic patches develop normally (111). Some studies have suggested that exogenously provided AHR agonists, such as tryptophan derivatives added to the diet, play a role in expanding and/or maintaining ILC3s (106, 112). Recent work has also shown that AHR agonists derived from commensal bacteria, such as lactobacilli, increase IL-22 production by ILC3s in the context of a tryptophan rich diet and this, in turn, confers resistance to infection by the opportunistic fungus Candida albicans (113). It is also possible that endogenous AHR ligands, such as kynurenine, produced by the enzyme tryptophan dioxygenase (TDO), may play a critical role in ILC3 biology (114).

Unexpectedly, AHR, in ILC3 cells, restrains adaptive Th17 responses. In AHR-deficient mice that lack ILC3s, Th17 cells in siLP undergo an uncontrolled expansion that leads to tissue damage. This Th17 proliferation is due to a dysregulation in the intestinal microbial niche that allows excessive growth of segmented filamentous bacteria (SFB), commensals known to drive Th17 differentiation (115).

In addition to RORγt and AHR, ILC3s also depend on Notch signaling for their development (111, 116). In RBP-J_k^−/− mice that lack all Notch signaling, NCR⁺ ILC3s are selectively reduced. Notch also promotes RORγt⁺ ILC differentiation from adult bone marrow precursors (117). Recent studies have also shown that NCR⁺ ILC3s, which develop post-natally and reach maximal expansion 4–6 weeks after birth in mouse small intestine LP (118), express Tbet (encoded by Tbx21) and require Tbet for development (46, 116, 119, 120). Induced by IL-23 and cues from the microbiota, Tbet is essential to instruct IFN-γ production and protection from pathogens such as S. typhimurium (46). Tbet expression seems to induce Notch, as Tbet haplo-insufficiency results in reduced Notch expression (116). However, it is conceivable that AHR and Tbet may cooperate in inducing Notch and final maturation of NCR⁺ ILC3s.

The requirement for Tbet in NCR⁺ ILC3s is somewhat surprising since human tonsil ILC3s do not express Tbet ex vivo (44). In fact, in steady state conditions, Tbet expression is limited to ILC1s and cNKs, while RORγt is selectively expressed by ILC3 (9).

Human tonsil ILC3s also express high levels of CCR6 and can easily be sorted at high purity based on NKp44 and CCR6 expression (44, 121), while mouse NCR⁺ ILC3s are CCR6 negative (46). Whether the discrepancies between the mouse and human system are linked to tissue specific differences (intestine versus tonsil) still remains to be determined. It is conceivable that Tbet may be needed at later stages of ILC3s differentiation, which may be poorly represented in the tonsil. In agreement with this hypothesis, tonsil ILC3s are characterized by an intrinsic plasticity and, when stimulated with cytokines such as IL-2 and IL-15 in vitro, can generate IFN-γ producing pro-inflammatory cells that acquire some features of cNK (8, 44). Specific engagement of cell surface receptors expressed by ILC3s by cognate ligands present in some tissues but not in others may also be an issue. Indeed, a recent study has suggested that stimulation of ILC3s via the DAP12-associated activating receptor NKp44 induces production of TNF-α and convert them to a more inflammatory-prone cell type (122). Experiments in mice have suggested that ILC3 conversion to an IFN-γ/TNF-α secreting pro-inflammatory cell type can occur in vivo (45). Moreover, recent fate-mapping experiments, aimed to track cells that expressed RORγt at any stage of their life cycle, show that “plastic” or “converted” ILC3s are present within the RORγt⁻NKp46⁺NK1.1⁺ population in siLP and cluster with siLP ILC1s and cNK (10).

Finally, STAT3 expression in ILC3s is required for robust IL-22 production and protection by intestinal infection with C. rodentium (123).

Group 2 ILCs necessitate RORα (124), Gfi1 (125), and Gata-3 for their development (94, 125–127). Gfi1 specifically controls the response to IL-33 and TSLP and the production of IL-5, but not IL-13, in ILC2s (125). The requirement for Gata-3 by ILC2s closely resembles the need for Gata-3 in Th2 differentiation. However, Gata-3 is also necessary for thymic NK cell development (26). Moreover, a recent study has indicated that Gata-3 is required for the generation of ILC3s (128), before the identification of the CHILP (10). Transfer of Gata-3-deficient fetal liver precursor cells in mice lacking ILCs prevents reconstitution of all RORγt⁺ ILC3s. This finding has led to the hypothesis that a common precursor might exist between ILC2s and ILC3s (128). In agreement with this view, another transcription factor TCF-1 (encoded by Tcf7) is required for ILC2s and NCR⁺ ILC3s (129, 130). TCF-1 in ILC2s acts downstream of Notch and forced expression of TCF-1 can bypass Notch requirement for ILC2 generation. TCF-1 also induces Gata-3, which is responsible for upregulation of Il-17rb (the receptor for IL-25) and Il2ra (CD25) in developing ILC2s. However, in the absence of Gata-3, TCF-1 directly controls the expression of Il7r (CD127) (130). In NCR⁺ ILC3s, TCF-1 acts downstream of Tbet and Notch to promote ILC3 development (129).

Gata-3 is also required to induce differentiation and/or maintenance of siLP ILC1s (10), as deletion of Gata-3 in NKp46⁺ cells results in a dramatic reduction of this ILC subset, while does not affect ILC3s or cNKs. In addition, early deletion of Gata-3 in hematopoietic cells abolishes the development of all CD127⁺ ILCs (131).

While it is not clear whether TCF-1 is also required for siLP ILC1s, the data available suggest that some transcription factors may be required at multiple stages of ILC differentiation and may be turned on and off at different transitional steps with a hierarchy that is still poorly defined.

Intraepithelial ILC1s and cNKs cells share some requirements in transcription factors for their differentiation. To begin, both cell populations require Tbet for development (9, 132). In addition, Tbet is necessary for the development of liver-resident NK cells that express Trail, CD49a, lack DX5, and Eomes (133) and have features of tissue-resident memory cells based on parabiosis experiments (30, 134). Although these cells were originally thought to represent an NK immature cell subset (135), more recent data suggest that they represent a distinct lineage of NK cells that share part of their transcriptional program with NKT cells (133). While it is not clear whether intraepithelial ILC1s derive from the CHILP, liver-resident NK cells could represent a true subset of ILC1s related to the siLP ILC1s, since they appear to differentiate from the CHILP (10).

Intraepithelial ILC1s and cNK cells also require E4BP4 (encoded by Nfil3) to develop (9, 136, 137). E4BP4 in NK cell development was previously thought to act directly downstream of IL-15R, which is required for NK cell maintenance (138, 139). However, more recent data suggest that viral infections can rescue NK cell development independently of E4BP4 in a process that requires inflammatory cytokines such as type 1 interferon and IL-12 along with ITAM signaling (140). E4BP4-independent NK cells are functional and persist in the periphery in an IL-15 dependent fashion. Ablation of E4BP4 after the earlier steps of NK cell commitment completely bypasses the requirement for E4BP4 in NK cell development (140). In addition, E4BP4 is less stringently required for NK cell development outside of the bone marrow. Liver-resident Trail⁺, DX5⁻ NK cells develop in the absence of E4BP4, especially when T cell competition is eliminated (30, 141, 142). Salivary gland NKs also do not require E4BP4 (29). The role for E4BP4 in thymic NK cell development is still controversial. Thymic NK cells seem to develop normally in vitro from E4BP4-deficient double negative (DN)1 and DN2 thymocytes (141). However, a second study indicates that thymic NKs in vivo develop along an E4BP4-dependent pathway (142). According to this report, E4BP4 is necessary to enforce Eomes expression in cNKs as well as in thymic NKs. Further confirming that E4BP4 may be necessary to drive Eomes expression, another report demonstrates that E4BP4 is strictly required for the transition from CLP to NK cell progenitor and regulates Eomes and Id2 expression in NK cells (143). Challenging the previous concept that E4BP4 acts downstream of the IL-15R (136), this study shows that E4BP4 operates upstream of IL-15 signaling and in the absence of E4BP4 CD122⁺ NK precursors fail to develop. E4BP4 expression is detected earlier that Tbet, Eomes, or Id2 expression and chromatin immunoprecipitation experiments demonstrate that E4BP4 promotes transcription of Eomes and Id2 by binding directly to the regulatory regions of their genes (143). These data are, however, in conflict with the study by Seillet et al., which shows that Id2 expression is normal in the absence of E4BP4 (142). Further studies will be necessary to elucidate the exact hierarchy in which transcription factors need to be expressed in order to grant successful development and maturation of NK cells and ILCs.

Finally, cNK cells share with CD4⁺ LTi-like cells the requirement for the HGM-box transcription factor superfamily member TOX. TOX-deficient animals lack NK cells and do not develop lymph nodes nor PP (144). Although it has been hypothesized that TOX controls induction or maintenance of Id2 in precursor cells, the exact mechanisms by which TOX regulates development of cNKs and LTi-like cells and whether it is necessary for other ILC subsets is unclear.

Studies over the past few years have indicated that adaptive immunity has adopted modules already established in the innate branch of the immune system to counteract pathogens and to maintain homeostasis at mucosal surfaces, which are constantly exposed to environmental insults and non-self-microbial communities. The identification of innate subsets that mimic Th1, Th2, and Th17/Th22 Thelper cells has greatly advanced our understanding of how immune responses are orchestrated and shaped. Future challenges in the field will be to understand whether additional subsets of ILCs are present that prevent or control autoimmune processes, i.e., whether an innate functional equivalent of regulatory T cells is in place. In addition, it will be important to delineate whether dedicated subsets of APCs or stromal cells are located in mucosal tissues that can preferentially activate a particular ILC group, or whether this activation process depends on specific signals provided by the intruder. Elucidating whether diverse inflammatory milieus can elicit different responses and immunological outcomes from the same ILC group will be also important. In the long term, a better understanding of the very first events that take place early on during the initiation of an immune response may be key in designing better strategies for vaccine development and therapeutic intervention.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.