REVIEW ARTICLE published: 04 March 2013 doi: 10.3389/fimmu.2013.00022 Diversity, function, and transcriptional regulation of gut innate lymphocytes

Lucille Rankin1,2†, Joanna Groom1,2†, Lisa A. Mielke1,2, Cyril Seillet1,2 and GabrielleT. Belz1,2* 1 Division of Molecular Immunology, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia 2 Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia

Edited by: The innate immune system plays a critical early role in host defense against viruses, Eric Vivier, Centre d’Immunologie de bacteria, and tumor cells. Until recently, natural killer (NK) cells and lymphoid tissue Marseille-Luminy, France inducer (LTi) cells were the primary members of the innate lymphocyte family: NK cells Reviewed by: form the front-line interface between the external environment and the adaptive immune Stephen K. Anderson, National Cancer Institute-Frederick, USA system, while LTi cells are essential for secondary lymphoid tissue formation. More Jacques Zimmer, Centre de recently, it has become apparent that the composition of this family is much more diverse Recherche Public de la Santé, than previously appreciated and newly recognized populations play distinct and essential Luxembourg functions in tissue protection. Despite the importance of these cells, the developmental *Correspondence: relationships between different innate lymphocyte populations remain unclear. Here we Gabrielle T. Belz, Division of Molecular Immunology, The Walter and Eliza Hall review recent advances in our understanding of the development of different innate Institute of Medical Research, 1G immune cell subsets, the transcriptional programs that might be involved in driving fate Royal Parade, Parkville, Melbourne, decisions during development, and their relationship to NK cells. VIC 3052, Australia. e-mail: [email protected] Keywords: innate lymphocytes, lymphoid tissue inducer cells, progenitor cells, transcription factors, differentiation, †Lucille Rankin and Joanna Groom are lymphoid tissue, immune protection equal first authors.

INTRODUCTION names have been applied to similar populations by the various Mucosal surfaces of the body are constantly bombarded with a research groups. As the diversity within the subsets has emerged, variety of both innocuous and pathogenic organisms. Lympho- the need for consistent nomenclature has become apparent (Spits cytes of the gut-, bronchus-, and nasal-associated lymphoid tissues et al., 2013). It is now suggested that ILCs be grouped into three (GALT, BALT, and NALT, respectively) play a critical role in pro- broad populations based on their phenotype, function, and tran- tecting the body from harmful pathogens that enter through the scriptional regulation (Tait Wojno and Artis, 2012; Spits et al., mucosal tissues and lung. 2013; Figure 2). Group 1 ILCs (ILC1) are composed of the proto- Innate lymphoid cells (ILCs) are an expanding family of typical ILC, natural killer (NK) cells. This classification, however, lymphocytes with innate cell characteristics. They co-ordinate might also include other ILCs that express the transcription fac- the organization of lymphoid tissues, maintain epithelial tissue tor T-bet and produce interferon-γ (IFN-γ; Spits and Di Santo, integrity, are responsible for the anatomical containment of com- 2011; Spits and Cupedo, 2012). Group 2 ILCs (ILC2) produce the mensal bacteria and play important roles in the protection against Th2 type cytokines interleukin (IL)-5 and IL-13 and are impor- pathogens early during infection. Individual populations of ILCs tant for helminth expulsion. The group 3 ILCs (ILC3) produce display distinct cytokine signatures in a manner analogous to the Th17 type cytokines IL-17 and IL-22. They comprise the clas- specialization of cytokine secretion found in T helper (Th) cell sical lymphoid tissue inducer (LTi) cells that are responsible for subsets (Figure 1) which has come to define ILC subsets as innate the generation of lymphoid tissue during embryogenesis, LTi-like versions, or perhaps as ancestors, of adaptive Th cells. Importantly, cells that are phenotypically similar to LTi cells but are enriched innate lymphocytes produce their effector cytokines in response in the intestine of the adult, together with the natural cytotoxicity to non-specific danger signals much more rapidly than their adap- receptor (NCR)-expressing NKp46+ ILC that produces IL-22 and tive T cell counterparts allowing them to act immediately, before not IL-17. antigen specificity is acquired. This rapid induction of effector Within these groupings, ILC subsets share some overlapping function allows ILCs to provide front-line protection at the onset features in their surface receptor phenotype and cytokine pro- of an immune response thereby limiting pathogen spread and duction but are distinct in their requirements for specific different regulating homeostatic tissue integrity. transcription factors. Specification of immune cell fate is a stepwise The heterogeneity of ILCs has become increasingly complex. process encoded by transcription factors that act on committed Initially, these cells were categorized based on their dominant multi-potent progenitors and sequentially restrict their develop- expression of a single cytokine and confusingly, many different mental fate to a particular lineage allowing them to develop into the mature cell type. Much has now been done in defining the regulatory circuits of B cells (Nutt et al., 2011), T cells (Kaech and Abbreviations: CP, cryptopatch; IL, interleukin; ILC, innate lymphoid cell; ILF, isolated lymphoid follicle; LN, lymph node; NCR, natural cytotoxicity receptor; NK Cui, 2012), NK cells (Sun and Lanier, 2011; Bezman et al., 2012), cell, natural killer cell; Ror, retinoic-related orphan. and dendritic cells (DCs; Belz and Nutt, 2012), but the lineage

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FIGURE 1 | Parallels between innate lymphocyte populations and CD4+ subset. As the different populations of innate lymphocytes have emerged, it helperT cell subsets. Multiple populations of CD4+ helper T cells exist and has become increasingly clear that these subsets produce an array of these are characterized by the production of signature cytokines by each cytokines that parallel those of the helper T cell subsets. defining transcription factors required for the differentiation of knowledge of the transcription factors that regulate the features of innate lymphocyte subsets is less well understood. Understanding ILCs (Figure 2; Table 1). the transcription factors and transcriptional networks that control the differentiation of innate immune cell populations is a rapidly EARLY DEVELOPMENT OF INNATE LYMPHOCYTES developing area of research. The progenitors of innate lymphocytes develop first from com- All ILC subsets are dependent on the transcriptional regula- mon lymphoid progenitors (CLPs) in the fetal liver (Sawa et al., tor inhibitor of DNA binding 2 (Id2) while ILC3 cells or Rorγt+ 2010; Vonarbourg et al., 2010) which give rise to multiple lym- ILCs rely on retinoic acid-related orphan receptor (Ror)γtdown- phoid lineages including B cells, T cells, and ILCs. In the fetal liver stream of Id2. A number of other transcription factors such as α4β7+ CLPs are able to differentiate into NK cells and all subsets Rorα, nuclear factor IL-3 [Nfil3, also known as E4-binding pro- of Rorγt+ ILCs. The development of ILCs from these precursors tein 4 (E4BP4)], thymocyte selection-associated high-mobility is dependent on the expression of Notch, which is regulated differ- group box protein (Tox), aryl hydrocarbon receptor (Ahr), ently in fetal and adult precursors (Possot et al.,2011). α4β7+ CLPs Runt-related transcription factor (Runx)/CCAAT-binding factor are severely reduced in Id2-deficient mice indicating that Id2 lies (Cbfβ), Gata-binding protein 3 (Gata3), and Notch have also been upstream of Rorγt in the developmental pathway (Cherrier et al., implicated in the development, survival, and function of these 2012). These cells also express the chemokine receptor CXCR6 populations. but this marker is not exclusive to LTi cells (Possot et al., 2011). As yet, the relationships between highly similar ILC populations It is proposed that both CD4+ and CD4− LTi cells arise from such as the different subsets of Rorγt+ ILCs and whether develop- aRorγt+α4+β7+ progenitor while NKp46+ ILCs emerge pre- mental plasticity enables dynamic inter-conversion between ILC dominantly from a Rorγt+α4+β7− cell fraction (∼50% NKp46+ subsets in response to environmental stimuli is unclear. Never- ILCs) suggesting they are distinct lineages (Narni-Mancinelli et al., theless, the recent rapid progress of this research area promises a 2011). Challenging this concept, it has also been reported that rich understanding of the complexity of the innate defense sys- Rorγt+ NKp46+ ILCs can arise from LTi cells following adop- tem protecting the body’s surfaces. Here we review the current tive transfer (Vonarbourg et al., 2010). Recently, our group has

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FIGURE 2 |Transcriptional programs controlling the development of without passing through a Rorγt-dependent stage. ILC2, however, rely on innate lymphocyte populations from their progenitor cells in the fetus Rorα and Gata3 for their development, expansion, and survival following and adult. The transcription factor Id2 is critical for the development of all NK stimulation. CD4+ and CD4− Id2+Rorγt+ LTi cells undergo diversification to cells and other ILC populations and is likely to direct the emergence of the generate terminally differentiated NKp46+ ILCs. CD4+ LTi cells appear to common innate lymphoid progenitor (CILP) downstream of the common have already established a stable phenotype. In contrast, CD4− LTi cells lymphoid progenitor (CLP) that does not express Id2. The first expression of expand in response to various cues such as stimulation through AhR. Further Id2 from cells derived from the CLP marks the emergence of the pre-pro NK environmental cues induce expression of T-bet and Notch ligands induce cells, the earliest progenitor of the NK cell lineage. LTi cells and NKp46+ ILCs Notch signaling which results in the development of NKp46+ ILCs that all express and are dependent on Rorγt while ILC2 and NK cells develop produce abundant IL-22 and protect against bacterial infection. discovered that NKp46+ ILCs arise solely from the CD4−Rorγt+ Natural killer cells, in contrast to LTi cells and NKp46+ ILCs ILCs and not the CD4+ LTi subset (discussed in more detail below; do not require Rorγt or IL-7 for their development. Until recently, Rankin et al.,2013). Thus, we provide evidence that CD4− LTi cells the earliest committed precursor of NK cells, the NKP,was identi- and NKp46+ ILCs are of the same lineage distinct from CD4+ LTi fied through the lack of expression of pan-NK cell markers such as (Rankin et al., 2013). This Rorγt+α4β7− subset which gave rise NK1.1 and CD49b and the expression of IL-2Rβ (Rosmaraki et al., to ∼50% NKp46+ cells was unable to generate CD4+ LTi cells 2001). Our recent development of an Id2-GFP (green fluorescent but could make CD4− Rorγt+NKp46− (Sawa et al., 2010). There- protein) reporter mouse where GFP is expressed under the control fore, it may be that this progenitor is the true CD4−LTi/NKp46+ of the endogenous Id2 promoter identified that this population ILC precursor that colonizes that gut while Rorγt+α4β7+ was heterogeneous and only a small fraction of cells expressed cells generate classical LTi cells essential for lymphoid tissue Id2 (Carotta et al., 2011; Jackson et al., 2011). This population generation. (lin−CD122+NK1.1−CD49b−Id2-GFP+) gives rise exclusively to

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Table 1 | Requirement of different transcription factors for lymphoid tissue and ILC development.

Transcription NK cells (ILC1) Nuocytes/natural LTi cells (NCR− ILC3) NKp46+ ILCs Lymphoid tissue organogenesis factor helper cells (ILC2) (NCR+ ILC3)

Id2 −−− −None (Yokota et al., 1999) RORγt ++− −Still have NALT (Eberl et al., 2004) RORα +−+ +Normal (Wong et al., 2012) Nfil-3 (E4BP4) −++ +Normal (Gascoyne et al., 2009) Gata-3 Lack thymic NK cells −+ +Normal (Hoyler et al., 2012; Mjosberg et al., 2012) AhR + ? CD4− LTi cells reduced Reduced No ILFs and CP Normal PP and LN (Lee et al., 2011) Tox Fail to mature ? Reduced Reduced No LNs; reduced size and number PPs >90% reduced ILCs (Aliahmad et al., 2010) Runx/Cbfβ2 + ? Reduced ? Impaired organization Reduced (Tachibana et al., 2011) T-bet Immature ++ – Normal

NK cells and has been defined as the pre-pro NK cell. This pop- et al., 1999; Hacker et al., 2003; Cannarile et al., 2006; Jackson et al., ulation has also more recently been identified using the surface 2011; Rankin and Belz, 2011). Genetic ablation of Id2 in mice marker CD244 and CD27 (Fathman et al., 2011). The induction results in the complete failure to develop lymph nodes (LNs), of Id2-GFP in the pre-pro NK cell identifies the first commitment Peyer’s patches, and other secondary and tertiary lymphoid tis- of progenitors to an NK but not T or B cell lineage. It will be sues including the NALT which is not affected by loss of other important to determine the identity of the common innate lym- transcription factors such as Rorγt(Yokota et al., 1999). Loss of phoid progenitor (CILP) and position it in the developmental tree the ability to form these lymphoid tissues has been attributed to to gain a greater understanding of the Id2-dependent precursors the lack of LTi cells in these mice although multiple ILC popula- of ILCs. tions are absent and could impact on development (Yokota et al., 1999; Cherrier et al., 2012). Id2−/− mice also express a marked INHIBITOR OF DNA BINDING 2 reduction in immature NK (iNK) cells in bone marrow and spleen Inhibitor of DNA binding 2 is a helix-loop-helix (HLH) transcrip- (Boos et al., 2007). In this setting, Id2 acts to inhibit the tran- tional regulator that plays diverse roles in directing lymphocyte scriptional program induced by E proteins and directs progenitors development and function. It is part of a group of closely related toward an innate cell lineage as deletion of E2A was able to partially proteins, Id1–4, that all share the highly conserved HLH motif overcome the need for Id2 in LTi cell development and restore the (Sun et al., 1991; Voronova and Lee, 1994). They regulate tran- development of lymphoid tissue. In addition, NK cell develop- scription by inhibiting the function of E-box proteins. E-box (or E) ment in the bone marrow was also restored, however, splenic NK proteins are another class of transcription factor and include the cells were poorly rescued, with only a small number of residual family members HEB, E2-2, and the E2A gene products E12 and cells which exhibited a thymic rather than splenic NK cell pheno- E47 (Murre et al., 1989a,b). E proteins are defined by their two type (Boos et al., 2007). Whether expression of Id2-GFP in an early highly conserved domains: (i) a HLH domain which regulates progenitor also marks the proposed CILP similar to the pre-pro homo- or heterodimerization and (ii) a basic domain is impor- NK cell remains unclear. tant for binding to E-box sequences on the DNA of target genes (CANNTG; Murre et al., 1989b). Id proteins are able to inhibit Runx/Cbfβ COMPLEX E protein function by forming a heterodimer with their comple- The Runx/Cbfβ complex has been shown to be involved in the mentary E protein via their common HLH domains. As Id proteins early differentiation pathway of LTi cells (Tachibana et al., 2011). lack the basic domain of E proteins, the Id/E protein heterodimer The Runx complex consists of a DNA binding subunit, Runx1, is unable to bind to DNA and thus transcription is blocked (Sun 2, or 3 and a non-DNA binding partner Cbfβ that increases the et al., 1991; Voronova and Lee, 1994). In addition to binding to E affinity of the Runx subunits for DNA. Runx is essential for the proteins, Id proteins have also been shown to interact with other differentiation of several hematopoietic cell lineages including B transcription factors including retinoblastoma protein (Rb), the cells, NK T cells, and CD8+ T cells (Okuda et al., 1996; Ichikawa ETS (E-twenty six) and Pax (Paired Box) families (Iavarone et al., et al., 2004; Egawa et al., 2005; Setoguchi et al., 2008). Mice lacking 1994; Yates et al., 1999; Roberts et al., 2001). either Runx1 or Cbfβ have defects in lymphoid tissue organo- Inhibitor of DNA binding 2 regulates a diverse number of cell genesis (Tachibana et al., 2011). Runx1/Cbfβ complexes regulate fate decisions during lymphopoiesis. It is involved in the develop- LTi cell differentiation at distinct stages prior to and following ment of effector CD8+ T cells, DCs, NK cells, and ILCs (Yokota Rorγt expression. In both Runx and Cbf-deficient fetuses, there

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was a significant reduction in the frequency of lin−α4β7+IL- mutant of NKp46 (Noé mice) has shed light on the mechanism 7RhighRorγt+ LTi precursor cells, a phenotype that parallels that of action of NKp46 (Narni-Mancinelli et al., 2012). Noé mice observed in Id2 and Rorγt-deficient mice (Tachibana et al., 2011). exhibit a point mutation (W32R) that prevents NKp46 expres- In contrast to Id2 and Rorγt loss, an even earlier, more pluripotent sion on the surface, but did not impair intracellular expression. precursor, the lin−α4β7+IL-7RintRorγt− population is reduced This phenotype was accompanied by increased responsiveness to in the absence of Cbfβ. Thus, Runx1/Cbfβ2 complexes impair stimuli and greater resistance to MCMV and influenza infection. LTi cell development prior to the induction of the Id2 and Rorγt Detailed molecular analyses showed that down-regulation of NK transcriptional programs. Given this finding, it might be expected cell activity was associated with silencing of the transcription fac- that NK cell development would also be significantly affected by tor Helios in the NK cells and attenuation of T cell responses. loss of Runx1/Cbfβ complexes. However, NK cells appeared nor- These studies revealed that NKp46 acts in a dose-specific man- mal in the absence of Runx1/Cbfβ, most likely because NK cells ner to tune the optimal development of the adaptive immune and CD8+ T cells dominantly express Runx3, rather than Runx1 response. (Ohno et al., 2008). In contrast, Cbfβ is required for NK cell devel- Murine NK cells require IL-15 signaling through the IL-15R opment (Guo et al., 2008). In the absence of Cbfβ, lin− fetal liver (composed of three subunits, namely IL-15Rα, CD122, and cells produce very few CD122 (IL2Rβ)+ cells capable of respond- CD132) for their survival (Mrózek et al.,1996; Kennedy et al.,2000; ing to IL-15 which is necessary to generate NK cells. CD122, a Cooper et al., 2002; Ranson et al., 2003). Although the majority of subunit of the IL-15R, is directly regulated by Cbfβ (Ohno et al., NK cells are generated in the bone marrow from CLPs, a devel- 2008). Nevertheless, transplantation of Cbfβ-deficient bone mar- opmentally distinct subset of NK cells that arise from bi-potent row into irradiated recipient adult C57BL/6 mice revealed that NK/T cell progenitors in the thymus (Vosshenrich et al., 2006). NKP cells could be detected in spleen indicating that NK defi- Bone marrow and thymic-derived NK cells can be clearly differ- ciency occurred at the NKP to iNK cell transition, whereas in entiated as thymic NK cells require the transcription factor Gata3 fetal liver, NKP could not be detected and thus the block in for development and are dependent on IL-7, in addition to IL- NK development in the fetus must occur prior to the emergence 15, for survival (Vosshenrich et al., 2006). They have an immature of NKP. phenotype with reduced cytotoxic activity and express lower lev- els of inhibitory receptors including the Ly49 family and CD94 NATURAL KILLER CELLS (ILC1) (Vosshenrich et al., 2006). In the bone marrow, multiple stages of Natural killer cells form part of the immediate response to NK cell development have been defined on the basis of their phe- pathogens or antigens expressed by transformed (i.e., cancer- notype, function, and proliferative capacity (Hayakawa et al.,2006; ous or stressed) cells and thus are critical during the innate and Huntington et al., 2007). These subsets can be distinguished by the adaptive immune responses (Vivier et al., 2011). They are distinct expression of CD27, CD11b (Mac-1), IL-7R, and KLRG-1, which from adaptive cells as they do not express somatically re-arranged are differentially regulated through maturation (Brady et al., 2004; antigen-specific surface receptors but instead respond through Hayakawa et al., 2006). iNK cells express low levels of CD11b and a repertoire of activating and inhibitory receptors (for example, the inhibitory receptors CD94 and the Ly49 family, do not express Ly49 molecules in mice, and killer cell immunoglobulin-like recep- CD49b (DX5) and exhibit low cytotoxicity (Rosmaraki et al., 2001; tors (KIRs) in humans; Raulet et al., 2001; Lanier, 2005). The Kim et al.,2002). During maturation, NK cells up-regulate CD49b, balance between stress-induced activation of NK cell receptors acquire Ly49 and CD94-NKG2 receptor expression then expand (such as the natural cytotoxicity triggering receptor 1, NCR-1, to become Mac-1high (Kim et al., 2002). Once they mature, NK encoding NKp46) and major histocompatibility complex class cells migrate to different tissues including the spleen, LNs, liver, I (MHC class I)-mediated inhibition controls the cytotoxicity lung, and skin where they develop tissue-specific phenotypes and of NK cells during pathogen infection and in destruction of functions. Furthermore, a unique subset of hepatic NK cells has tumors. been described, which constitutively expresses Trail and IL-7R but Natural cytotoxicity receptors are immunoglobulin-like trans- not Eomesodermin (Eomes) develops independently of the bone membrane glycoproteins which signal through adaptor proteins marrow (Andrews and Smyth, 2010) and persists as a stable lin- with intracellular tyrosine-based activation motifs (ITAMs). They eage in the liver (Takeda et al., 2005). However, the ontogeny and are mostly expressed on NK cells. Humans can express NKp30, specific functions of these unconventional NK cells is yet to be NKp44, and NKp46 while mice express NKp46 exclusively (Spits fully elucidated. and Cupedo, 2012). Gazit et al. (2006) adopted an elegant Natural killer cells found in the intestinal environment are approach to investigate the role of NKp46 in NK cells by insert- present at low frequencies. Functionally and transcriptionally, they ing GFP into the Ncr1 locus. This enabled direct visualization of are more similar to other conventional NK (cNK) cells than the NKp46 expression on NK cells during influenza infection where intestinal ILCs (Reynders et al., 2011). In the intestinal lamina pro- NKp46 recognizes the ligand viral hemagglutinin in a sialic acid- pria, a large proportion of NK cells express the markers IL-7R and dependent manner (Mandelboim et al., 2001). It also showed that c-Kit and do not express Eomes, a phenotype shared by immature NKp46+ NK cells that accumulated in the lung were critical for cNK cells, thymic NK cells, and liver-derived NK cells. In addition, the control of lethal influenza. Using this model it has also been an unusually high proportion of these NK cells lack the expression demonstrated that NKp46 expression by NK cells is important in of CD27 and maturation markers such as KLRG-1 and CD11b. controlling tumors (Gazit et al., 2006) and for the development Further phenotypic analysis of intestinal NK cells has shown they of type-1 diabetes (Gur et al., 2010). Recently, a loss-of-function express only low levels of NKG2D, the Ly49 receptors and exhibit

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low cytotoxic activity, again indicating their immaturity (Sanos cells with decreased cytotoxic activity (Barton et al., 1998). The et al., 2009). Whether this unique subset of intestinal NK cells are mechanism by which Ets-1 regulates the development and effector a distinct lineage or alternately are a gut-specific version of thymic function of NK cells has only recently been described. Ets-1 acts at or hepatic NK cells has not yet been addressed. Nevertheless, effec- the pre-pro NK stage, directly regulating the expression of Id2 and tor molecules such as perforin, granzymes, and IFN-γ produced by T-bet (Ramirez et al., 2012). The residual Ets-1−/− mature NK NK cells following activation play crucial roles in defense against cells have reduced expression of activating receptors like NKp46, tumors and viral infections. NK cells are therefore thought to rep- Ly49D, and Ly49H resulting in lower cytotoxic capacity (Barton resent the innate version of CD4+ Th1 cells and cytotoxic CD8+ et al., 1998). Interestingly, Ets-1−/− NK cells also exhibit increased T cells (Trinchieri, 1989; Sun and Lanier, 2011). expression of IL-15-regulated genes as well as inhibitory receptors, which is a characteristic of chronically activated NK cells. Thus, TRANSCRIPTIONAL REGULATION OF NK CELLS Ets-1 controls a broad range of transcription factors and receptors Nuclear factor interleukin-3 that drive NK cell development and function (Ramirez et al.,2012). Nuclear factor IL-3 is a critical transcriptional regulator for NK cell development, but does not appear to affect the development Tox of any other ILC subsets (Gascoyne et al., 2009; Kamizono et al., Tox, similar to Id2 and Ikaros, plays a role in the development 2009). The blockade in NK development occurs at the NKP to of both LTi and NK cells. Tox was firstly described as essen- iNK transition. Residual NK cells found in Nfil3−/− mice have tial for T cell development (Wilkinson et al., 2002; Aliahmad and −/− impaired cytotoxic activity and reduced IFN-γ production sug- Kaye, 2008) but subsequent studies have shown Tox mice to gesting that Nfil3 could also be essential for maintaining mature have severe defects in mature NK cells in the bone marrow and NK cell function (Gascoyne et al., 2009; Kamizono et al., 2009). peripheral lymphoid tissues (Aliahmad et al., 2010). Furthermore, Nfil3 is induced following IL-15 signaling through the IL-15R and knockdown or over-expression studies in human hematopoietic is thought to act prior to the requirement for Id2 in NK cell devel- stem cells have also shown Tox to be an important regulator of NK opment (Gascoyne et al., 2009). Indeed, over-expression of Nfil3 cell development in humans (Yun et al., 2011). In the few remain- −/− results in an increase in the expression of Id2 in hematopoietic ing Tox NK cells, Id2 expression was significantly reduced but −/− −/− progenitors and was sufficient to partially rescue NK cell pro- unlike in Nfil3 mice, ectopic expression of Id2 in Tox bone duction from progenitors. Thus, Nfil3 was thought to directly marrow precursors could not rescue NK cell development (Aliah- induce Id2 in the NK cells (Gascoyne et al., 2009). This conclu- mad et al., 2010). Thus, loss of Id2 expression cannot fully explain −/− sion assumes that Nfil3 and Id2 lie sequentially in the signaling the developmental defect observed in Tox mice, suggesting pathway which may not be the case, as loss of Nfil3 resulted in that additional regulatory pathways exist for the development of only ∼30% reduction in Id2 expression. An alternate possibility is NK cells. that Id2 and Nfil3 may exert separate critical effects in parallel in these cells. NUOCYTES, NATURAL HELPER CELLS, AND INNATE HELPER CELLS (ILC2) T-box factors T-bet and Eomes Over the last 3 years, a new family of innate lymphocyte has T-bet (encoded by Tbx21) and Eomes (encoded by Eomesoder- been identified that are able to initiate Th2-like immune responses min) are highly homologous T-box transcription factors that play (Neill and McKenzie, 2011). These have been referred to as nat- important roles in regulating the function of multiple cell types ural helper cells (Moro et al., 2010), innate helper cells (Price especially NK cells (Lazarevic and Glimcher, 2011; Gordon et al., et al., 2010) and nuocytes (Neill et al., 2010). These populations 2012). In particular T-bet, like the transcriptional repressor B are Rorα-dependent ILCs that express CD278 (ICOS), ST2 (IL- lymphocyte-induced maturation protein 1 (Blimp1) has been sug- 33R), and IL-17BR (Scanlon and McKenzie, 2012). Whether these gested to be involved in maturation and acquisition of effector ILCs represent tissue-specific versions of the same cell type is not functions in NK cells (Townsend et al., 2004; Jenne et al., 2009; yet understood, but they can collectively be referred to as ILC2s Kallies et al., 2011). More recently it has been shown that these due to their common production of T helper type 2 cytokines, IL- two transcription factors act in a sequential manner whereby T-bet 13, IL-5, IL-4, and IL-6 (Moro et al., 2010; Neill et al., 2010; Price directs the development of iNK cells and stabilizes the immature et al., 2010; Saenz et al., 2010). These cytokines can activate mast phenotype (Gordon et al., 2012). Eomes allows NK expression of a cells, basophils, and eosinophils, which are important for protec- diverse repertoire of Ly49 receptors and maintenance of a mature tion against parasitic infections, and have also been implicated in phenotype (Gordon et al., 2012). In addition, T-bet and Eomes the development of allergic diseases such as asthma (Neill et al., appear to have distinct roles in the emergence of different NK cell 2010; Neill and McKenzie, 2011). ILC2s expand and secrete these subsets. For example, deficiency of T-bet leads to the loss of hepatic potent Th2 cytokines in response to the IL-17 family member IL- NK cells but not cNK cells (Gordon et al., 2012), suggesting a spe- 25 and the IL-1 family member IL-33 (Moro et al., 2010; Neill cific developmental pathway for intrahepatic NK cells (Andrews et al., 2010). Nuocytes and natural helper cells are lin− IL-7R+ and Smyth, 2010). and express the stem cell markers c-Kit and Sca-1. Natural helper cells were first discovered in clusters of lymphocytes found in adi- Ets-1 pose tissue termed fat-associated lymphoid tissue (FALT; Moro Ets-1 is a member of the Ets family of winged HLH transcription et al., 2010) while nuocytes have been shown to be essential for the factors. Ets-1−/− mice display significantly reduced splenic NK expulsion of helminths during infection (Moro et al., 2010; Neill

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et al., 2010; Price et al., 2010; Saenz et al., 2010). An IL-33 respon- Rorα is critical for their terminal differentiation and their capacity sive population of ILC2s located in the lung also secrete IL-13 and to effect immunity against intestinal worms (Wong et al., 2012). IL-5. They regulate airway epithelial integrity and promote tissue This positions Rorα as an essential regulator of the ILC2 lin- remodeling following lung disease such as influenza virus infec- eage downstream from Id2. “Stagger” mice (Rorαsg/sg) exhibit tion (Monticelli et al., 2011). During the development of allergic a spontaneous deletion in Rorα that prevents translation of the lung disease IL-9 production further promotes secretion of IL-5 ligand-binding homology domain of Rorα and provide a novel and IL-13 in ILCs (Wilhelm et al., 2011). Interestingly, cytokine tool for investigating the biology of Rorα in the immune system production in this setting is dependent on collaboration with the (Hamilton et al., 1996). ILC2s are readily expanded in vivo by the adaptive immune response as IL-2 is required for ILC-derived IL-9 administration of IL-25. In the stagger mice, injection of IL-25 was production. In turn, these ILC2 cells promote the generation and unable to expand the nuocyte population in contrast to wild-type expansion of IL-13 producing T cells (Neill et al., 2010). Another littermate controls (Wong et al., 2012). Furthermore, loss of the significant population of IL-33 responsive ILC2s is also present nuocyte population in the lung mucosal tissues resulted in exacer- in the intestine of mice and most likely important for helminth bated responses to asthma, and in the gut, impaired immunity to expulsion (Hoyler et al., 2012). A human equivalent of the IL-13 helminthes infections such as N. brasiliensis. ILC has also been identified in nasal polyps of patients with chronic rhino-sinusitis suggesting a role for ILC2s in the pathology of type Rorγt-DEPENDENT INNATE LYMPHOID CELLS (ILC3) 2 immunity-associated diseases (Mjosberg et al., 2011). The Rorγt-dependent ILCs are divided into LTi cells, and a heterogeneous population of Rorγt+ ILCs found in the intesti- TRANSCRIPTIONAL REGULATION OF ILC2s nal mucosa. These mucosal Rorγt+ ILCs include NKp46+ ILCs Gata-binding protein 3 (NCR+ ILC3) and LTi-like cells (NCR− ILC3) populations. Recent evidence suggests that similarities in the transcriptional programs exist between ILC2 and Th2 cells. Gata3 is required for LTi CELLS AND LYMPHOID TISSUE ORGANOGENESIS the differentiation and maintenance of ILC2 and is the fate deter- Lymphoid tissue inducer cells were originally reported as an mining transcription factor for differentiation of Th2 cells (Hoyler obscure subset of CD4+CD3− lymphocytes able to colonize the et al., 2012). Gata3 belongs to the family of transcription factors mesenteric LNs and peripheral LN anlagen in the developing that bind DNA at the WGATAR motif (Orkin, 1992). It contains mouse fetus (Kelly and Scollay, 1992; Mebius et al., 1997). It is two Gata-type zinc fingers and regulates a number of important now known that they are comprised of a heterogeneous pop- aspects of T cell function including the secretion of IL-4, IL-5, ulation of cells where only a proportion express CD4 and they and IL-13 (Ho et al., 2009). Mice lacking Gata3 lack IL-13 pro- require signaling through IL-7R for their survival and expansion ducing cells, the majority of which are ILC2 cells that express high (Adachi et al., 1998; Yoshida et al., 1999; Finke et al., 2002; Kim levels of Gata3 (Moro et al., 2010; Halim et al., 2012; Liang et al., et al., 2003, 2008; Lorenz et al., 2003; Mebius, 2003; Eberl et al., 2012). These mice phenocopy IL-13-deficient mice and display 2004; Coles et al., 2006; Satoh-Takayama et al., 2010). They are significantly increased susceptibility to Nippostrongylus brasiliensis essential for the formation of lymphoid tissues including LNs and gut infection. While these observations highlight parallels between mucosal-associated lymphoid tissues (MALT) which encompasses Th2 cells and ILC2 cells, NK cells also rely on Gata3 for maturation, the NALT, GALT, and BALT. expression of homing molecules and production of IFN-γ (Sam- During fetal development, LTi cells colonize LN and Peyer’s son et al., 2003). Thus, Gata3 plays an important role in terminal patch anlagen where mucosal addressin cell adhesion molecule-1 differentiation and acquisition of full effector function of multi- (MadCam-1), the ligand for α4β7, is highly expressed (Mebius ple innate lymphocyte populations. However, through a cis-acting et al., 1996). They are the first hematopoietic cells recruited to  regulatory element located 280 kb 3 to the Gata3 gene that appears secondary lymphoid tissue where they use LTα1β2 to interact to be both T cell and NK cell-specific (Hosoya-Ohmura et al., with the LTβR expressed on mesenchymal cells at LN and Peyer’s 2011), therefore, Gata3 was postulated to play an important role in patch anlagen (Yoshida et al., 1999, 2002; Mebius, 2003). Acti- directing the differentiation of individual innate populations per- vated mesenchymal cells then express adhesion molecules as well haps through the expression of cell-specific enhancers. In parallel as pro-inflammatory cytokines and chemokines to enable recruit- with these observations it was discovered that human ILC2s also ment and organization of lymphocytes in a process similar to highly express Gata3 and this expression, together with enhance- inflammation. Tumor necrosis factor activation-induced cytokine ment of signal transducer and activator of transcription 5 (STAT5) (TRANCE) signaling is required for LTi cell accumulation at LN activation, was driven by expression of the cytokine thymic stro- anlagen leading to early embryonic development of peripheral mal lymphopoietin (TSLP; Mjosberg et al., 2012). These findings LNs, while the loss of IL-7R leads to an absence in develop- emphasize the complexity of the emerging transcriptional network ment of both LNs and Peyer’s patches (Adachi et al., 1998; Kim guiding ILC development, and in addition, underline the similar- et al., 2000). ities that exist in the regulation of these innate cell types between mouse and man. Rorγt+ ILCs IN THE INTESTINAL MUCOSA – LTi-LIKE ILCs (NCR− AND NCR+ ILC3) Rorα Subsets of Rorγt+ ILCs that are developmentally and function- Although ILC2s do not require Rorγt for development, it has ally similar to LTi cells but show distinct phenotypic differ- recently been shown that the highly related transcription factor ences have been described in the intestinal mucosa. These have

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been termed LTi-like cells. They include NCR− ILC3 which during intestinal inflammation and are potent inducers of colitis can be further divided into CD4−NKp46−CCR6+/−Rorγt+ (Vonarbourg et al., 2010). and CD4+NKp46−CCR6+Rorγt+ subsets, and NCR+ ILC3s + (CD4−NKp46+Rorγt+). Rorγt ILCs during inflammation These Rorγt+ ILCs are most highly represented in tertiary In addition to the key role Rorγt+ ILCs play in the develop- lymphoid structures of the GALT. CD4+ LTi cells are most promi- ment of lymphoid tissues in the intestinal mucosa, they are nent at birth and appear to be involved in the formation of crucial for maintaining epithelial integrity through the produc- Peyer’s patches. However, after birth, CD4+LTi only make up tion of IL-22 and IL-17, signature cytokines secreted by IL-17 15–20% of the NCR− ILC3+ populations when the CD4− LTi producing Th cells (Wolk et al., 2004; Ivanov et al., 2006; Zheng cells and NKp46+ ILCs rapidly expand at the same time as micro- et al., 2007; Pickert et al., 2009; Takatori et al., 2009; Vivier et al., bial colonization, indicating an important interplay between their 2009; Ota et al., 2011). ILCs are the main producers of IL- development and environmental signals. 22 in the intestine following stimulation by IL-23 produced by activated intestinal DCs (Sawa et al., 2011; Cox et al., 2012). Inter- LTi-LIKE ILCs (NCR− ILC3) IN THE GENERATION OF TERTIARY LYMPHOID estingly, at steady state the adaptive immune system represses TISSUE IL-22 production by ILCs, however this is de-repressed follow- Secondary lymphoid tissue of the GALT, namely mesenteric LN ing epithelial damage (Sawa et al., 2011). IL-22 activates the and Peyer’s patch develop in an organized and programed way transcriptional regulator STAT3 and triggers production of anti- involving fetal LTi cells, whereas tertiary lymphoid structures microbial molecules such as β-defensin, lipocalin-c and Reg family develop in response to the environmental cues from microbiota proteins from epithelial cells to limit pathogen survival and dis- and surrounding tissue and are thought to involve NCR− ILC3 semination (Liang et al., 2006; Sugimoto, 2008; Zheng et al., 2008; CD4− or CD4+ cells (Lorenz et al., 2003; Eberl et al., 2004; Ota Sonnenberg et al., 2011). IL-17 is a pro-inflammatory cytokine et al., 2011). Here, LTi-like cells are able to induce the develop- that promotes neutrophil recruitment and activation and is also ment of well-organized lymphoid structures capable of carrying enhanced by IL-23 (Takatori et al., 2009). In contrast to LTi cells, out germinal center reactions and promote immunoglobulin A murine NKp46+ ILCs are not known to produce IL-17 (Luci (IgA) class switching in response to inflammation (Lorenz et al., et al., 2009; Sawa et al., 2011) although the complete spectrum 2003; Tsuji et al., 2008). They are key effector cells that drive both of cytokines that this population does produce during inflam- the size and number of cryptopatches (CPs) and isolated lymphoid mation has not been fully assessed. Rorγt is known to regulate follicles (ILFs) in the gut tissues. IL-17, so it is intriguing that these Rorγt+ILCs lack production of this cytokine implying different transcriptional regulation of + NKp46+ ILCs (NCR+ ILC3) cytokines in NKp46 ILCs compared with LTi cells. The impor- A major subset of intestinal Rorγt+ ILCs express the surface tance of IL-23 induced IL-22 production by Rorγt+ ILCs is molecule NKp46 in addition to the transcription factors Rorγt demonstrated during experimental models of intestinal inflam- and Id2. NKp46+ ILCs rely on IL-7 signaling for survival and mation such as Citrobacter rodentium, as mice deficient in these are themselves potent producers of IL-22 but unlike NCR− ILC3s cells rapidly succumb to infection (Satoh-Takayama et al., 2008; do not produce IL-17 (Satoh-Takayama et al., 2008, 2010; Reyn- Zheng et al., 2008). Here, ILC-derived IL-22 can be induced by ders et al., 2011). NKp46+ ILCs reside mainly in CPs, but are also lymphotoxin-β in the presence of IL-23 and reduces the sys- found in ILFs, Peyer’s patches and at very low frequencies in the temic dissemination of bacteria from the colon to the liver and mesenteric LNs (Luci et al., 2009). NKp46+ ILCs can be further spleen (Ota et al., 2011; Sonnenberg et al., 2011). While IL-17 and divided according to their expression level of Rorγt and NK1.1 IL-22 are important to maintain intestinal homeostasis and pro- into RorγthiNK1.1lo/− and RorγtintNK1.1int populations, how- tect against intestinal pathogens, aberrant production of these ever whether these are distinct subsets remains unclear (Luci et al., cytokines has been associated with inflammatory bowel disease 2009). The human counterpart for NKp46+ ILCs are defined by and colon cancer. IL-17 produced by ILCs is thought to be their expression of NKp44 rather than NKp46 (Crellin et al.,2010). one of the key cytokines in driving colon inflammation in an NKp46 was originally presumed to be exclusively expressed by NK innate model of Helicobacter hepaticus driven colitis (Buono- cells. It was therefore assumed that NKp46+ ILCs were most closely core et al., 2010). Human studies show that IL-17 production related to NK cells, however, NKp46+ ILCs are distinct from NK and the frequency of ILCs increases in patients with inflamma- cells in several fundamental ways. NKp46 is not essential for the tory bowel disease (Geremia et al., 2011). IL-22 can also promote development or function of NKp46+ ILCs, which contrasts with intestinal disease by enhancing epithelial cell proliferation leading the important role of NKp46 in NK cells (Satoh-Takayama et al., to colon cancer (Huber et al., 2012). Therefore it is important 2009; Narni-Mancinelli et al., 2011). IL-15 is essential for the dif- to understand the activating factors and molecular pathways ferentiation, survival, and activation of NK cells but is not required controlling IL-17 and IL-22 production as this will eventually for the development of NKp46+ ILCs, which like LTi cells depend lead to the development of improved treatments for intestinal on the cytokine IL-7 (Satoh-Takayama et al., 2009, 2010). Func- disease. tionally, NKp46+ ILCs also differ from NK cells as they produce NKp46+ ILCs are a critical source of IL-22 but despite this, the little IFN-γ, no perforin and show little cytotoxic potential (Satoh- relative contribution of IL-22 secreted from NKp46+ ILCs or other Takayama et al., 2008; Reynders et al., 2011). An exception to this LTi-like cell subsets is not clear. Until recently, the extent to which is RorγtintNKp46+ ILCs that can be induced to produce IFN-γ one population might dominate or compensate for the other may

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depend on the type of mucosal activation was unclear, however we depleted in Rag−/− mice and a specific species of commensals have recently shown that other Rorγt+ cells cannot compensate for that reside in Peyer’s patches and mesenteric LNs named Alcali- the absence of IL-22 producing NKp46+ ILCs during C. rodentium genes disseminated to the liver and spleen. This resulted in systemic infection (Rankin et al., 2013). inflammation and increased levels of lipopolysaccharide (LPS). As such, ILCs were crucial for the specific anatomic containment of + Rorγt ILCs and commensal microflora Alcaligenes through the production of IL-22. During steady state, Rorγt+ ILCs play critical roles in maintaining mucosal homeostasis through responding to carefully regulated TRANSCRIPTIONAL REGULATION OF ILC3 signals from the epithelial layer and commensal bacteria in the Rorγt intestine. Firstly, the induction of ILFs from CPs occurs in response The orphan nuclear receptor Rorγt is an important transcrip- to external stimuli such as peptidoglycan from gram-negative bac- tion factor involved in lymphocyte development. It belongs to a teria in the gut lumen (Bouskra et al., 2008). In this situation, B large family of steroid hormone receptors that include receptors cells and other lymphocytes are recruited to CPs in a lympho- for steroids, retinoids, thyroid hormones, and vitamin D3 and toxin receptor-dependent manner (Lorenz et al., 2003; Bouskra are important regulators of development, cell differentiation, and et al., 2008). Interestingly, ILFs do not develop in germ-free mice, organ physiology (Mangelsdorf et al., 1995). Rorγtisencodedby but instead remain as CPs (Hamada et al., 2002; Pabst et al., 2006; the Rorc gene and encodes two isoforms, Rorγ and Rorγtfrom Bouskra et al., 2008). Therefore, the number of CPs, ILFs and distinct promoters (He et al., 1998; Villey et al., 1999). While the commensal microflora are tightly controlled by a dynamic acti- mRNA of the first isoform, Rorγ, is detectable in many tissues, the vatory/inhibitory negative feedback loop (Bouskra et al., 2008). expression of Rorγt is limited to a small number of lymphocyte Increased formation of ILFs enhances the capacity of the immune populations. Rorγt is highly expressed in double positive (DP) thy- system to target responses toward the luminal bacteria. In turn, mocytes (Villey et al., 1999; Sun et al., 2000). The thymus of mice this restricts bacterial proliferation and invasion and serves to shut deficient in Rorγt is dramatically decreased in size and cellularity down the signals that induce ILFs (Sawa et al.,2010; Ota et al.,2011; (Kurebayashi et al.,2000; Sun et al.,2000). This is mainly due to the Sonnenberg et al., 2011). Adult LTi cells become activated and reduced number of CD4+CD8+ DP thymocytes that results in the induce recruitment of other lymphocytes through a chemokine- reduction of mature single positive (CD4+CD8− or CD4−CD8+) mediated network. CCR6 responds to signaling from CCL20 (also thymocytes. Rorγt−/− thymocytes also show increased levels of known as MIP-3α), a molecule highly expressed by the epithelium apoptosis due to the failure of induction of the pro-survival pro- overlying Peyer’s patches and ILFs. β-defensin 3 also binds CCR6 tein Bcl-xL in the absence of Rorγt(Eberl and Littman, 2004). It and is expressed by inflamed epithelia and intestinal CPs. Thus, was therefore proposed that Rorγt prolongs the lifespan for DP it is suggested that Rorγt+ ILCs depend on CCR6 responses to thymocytes allowing them a greater chance to undergo positive CCL20 and β-defensin signaling (Sawa et al., 2010). selection (Kurebayashi et al., 2000). Beyond its role in thymocyte Secondly, Rorγt+ ILCs constitutively produce IL-22 where the development, Rorγt expression does not appear again in the T levels are controlled by careful integration of signals from com- cell development program until peripheral CD4+ Th cell sub- mensal bacteria and the epithelial layer. Vonarbourg et al. (2010) sets undergo differentiation. In this population, Rorγt is induced also showed that commensal microflora and IL-7 can act to sta- in response to IL-6 and transforming growth factor beta (TGF-β) bilize Rorγt expression within the NKp46+ ILC population, but signaling to drive naïve CD4+ T cells toward a Th17 fate character- they apparently do not induce their differentiation. Other reports ized by the production of IL-22 and IL-17 (Ivanov et al., 2006). In have shown that intestinal microbiota have no effect on relative or the absence of Rorγt, naïve CD4+ T cells are unable to differentiate absolute numbers of ILCs in the gut (Reynders et al., 2011; Sawa into Th17 cells. et al., 2011) while earlier reports showed a reduction in NKp46+ Similar to Id2-deficient mice, Rorγt null mice lack secondary ILCs, but not LTi cells in germ-free mice suggesting a role for com- lymphoid tissues due to the absence of LTi cells (Sun et al., 2000; mensal microflora in directing their development and function Eberl and Littman, 2003). This includes LNs, Peyer’s patches, (Satoh-Takayama et al., 2008; Sanos et al., 2009; Vonarbourg et al., ILFs, and CPs. However, in contrast to Id2-deficient mice, NALT 2010). It has now been reported that the absence of microflora development remains unperturbed in the absence of Rorγt(Eberl enhanced the ability of NKp46+ ILCs to produce IL-22 (Reyn- and Littman, 2003). Using the elegant Rorγ tGFP/GFP mouse strain ders et al., 2011; Sawa et al., 2011). The microbiota were shown to (which are deficient in Rorγt) it has demonstrated that both LTi induce IL-25 expression on epithelial cells which in turn inhib- cells and all subsets of Rorγt+ ILCs in the intestine are dependent ited NKp46+ ILC IL-22 secretion (Sawa et al., 2011). As IL-25R on Rorγt for their development while NK cells and nuocytes are is not expressed on Rorγt+ ILCs, this must be occurring via an unaffected (Eberl et al., 2004; Satoh-Takayama et al., 2008; Luci intermediate cell type such as intestinal DCs. While analyses of et al., 2009; Sanos et al., 2009; Moro et al., 2010; Neill et al., 2010; the contribution of microbiota to the development, function, and Vonarbourg et al., 2010). Recent microarray and fate mapping proliferation of ILCs has been investigated, virtually nothing is experiments on cells expressing the cell surface receptor NKp46 known about how the individual ILC populations might modify further support the notion that Rorγt is a lineage defining tran- the microbiome. scription factor for the all ILC3 cells as intestinal NK cells and Thirdly, in a recent elegant study, intestinal ILCs were shown NCR+ ILC3s were shown to represent two distinct lineages both to be crucial for the anatomical containment of commensal bac- genetically and developmentally (Narni-Mancinelli et al., 2011; teria during steady state (Sonnenberg et al., 2012). Here ILCs were Reynders et al., 2011).

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Tox Notch In addition to it’s essential role during NK development (above), Notch proteins (Notch1–4) are transmembrane receptors that Tox is also required for the development and maturation of innate bind to the surface ligands Delta-like or Jagged before induc- lymphocytes (Neill and McKenzie, 2011). Tox −/− mice have ∼ tion of proteolytic cleavage to release the Notch intracellular 90% reduction in LTi cells in the spleen of neonates and NKp46+ domain (ICN). On release, ICN translocates to the nucleus where ILCs are also diminished (Aliahmad et al., 2010). In contrast to it binds to the CSL/RBP-J (CBF-1/RBP-J, Suppressor of Hairless, mice that lack Id2, in the absence of Tox, Peyer’s patches still Lag-1) transcription factor displacing co-repressors and recruit- develop but their size and number are severely reduced. Over- ing co-activators of the Mastermind-like family (MAML). Notch expression of Id2 was unable to recover a normal phenotype in signaling in developing lymphocytes is complex (Bray, 2006). In the Tox −/− LTi cells suggesting that Tox is downstream of Id2. peripheral CD4+ T cells, Notch-1 plays an important role in However, such experimental approaches do not excluded that Id2 Th1 polarization (Minter et al., 2005). Both Notch-1 and Notch-2 and Tox may not lie in the same linear pathway, or that Tox may together with the canonical effector RBPJ (recombination sig- regulate other critical Id2-independent steps in ILC development. nal binding protein for the immunoglobulin kappa J region) act In addition, the role of Tox in specific subsets of intestinal ILCs, to protect activated cells against apoptosis (Helbig et al., 2012). including NKp46+ ILCs, were not investigated in detail in this In innate lymphocytes, Notch-2 was strongly implicated in the study. Nevertheless, although Tox and Id2 are both highly influ- development of adult, but not fetal, Rorγt+ cells due to the dif- ential in regulating the development of NK and LTi cells, Tox ferentiation of progenitors in response to the stromal cell OP9 appears to act later and differentially affects Peyer’s patches and expressing the Notch ligand DL-4 (Possot et al., 2011). Disruption LN formation (Aliahmad et al., 2010). of Notch signaling, by the deletion of RBPJ, reduced the num- ber of NK46+ ILCs in the small intestine though it had only a Aryl hydrocarbon receptor marginal effect on other Rorγt+ ILC subsets and thus ILF and CP Aryl hydrocarbon receptor is a ligand-dependent transcrip- development and this was thought to be downstream of Ahr (Lee tion factor that is a member of the basic HLH/Per-Arnt-Sim et al., 2011). The circuitry regulating the differential dependence (bHLH/PAS) family. AhR is a cytosolic transcription factor bound of Notch in different ILC subsets appears to depend largely on the to co-chaperones such as hsp90 and is normally inactive. On induction of T-bet (Rankin et al., 2013). ligand binding, it dissociates from its chaperones and translo- cates to the nucleus dimerizing with the AhR nuclear translocator T-bet (ARNT) that results in changes to gene transcription (Burbach The role of T-bet in the development of innate lymphocytes has et al., 1992). AhR is a sensor of a number of chemicals and not previously been investigated except in NK cells. Our recent environmental toxins including as 2,3,7,8-tetrachlorodibenzo-p- data showed that T-bet was highly expressed by both the Rorγt + dioxin (TCDD) and phytochemicals such as indole-3-carbinol intermediate and high populations of NKp46 ILCs but not the − + found in cruciferous vegetables such as cauliflower and cab- NKp46 Rorγt ILCs. Indeed, T-bet was found to be essential + bage. Endogenous ligands such as the tryptophan photo- for the differentiation of NKp46 ILCs (Rankin et al., 2013). Fur- − + product 6-formylindolo-3,2-b-carbazole (FICZ) have also been thermore, T-bet was crucial for the transition from CD4 Rorγt + + identified. LTi cells into Rorγt NKp46 ILCs and this transition depended Aryl hydrocarbon receptor has been shown to affect the differ- on Notch signaling. Interestingly, both ILC subsets were strongly − entiation of regulatory T cells (Tregs) and enhance the production affected by the absence of Ahr, indicating that CD4 LTi cells are + of IL-17 from Th17 cells. Furthermore, AhR is necessary to allow a lineage distinct from CD4 LTi cells (Kiss et al., 2011). Similar + Th17 cells to produce IL-22 (Kimura et al., 2008; Quintana et al., to NK cells, Blimp1 was also exclusively expressed by NKp46 2008; Veldhoen et al., 2009; Mezrich et al., 2010). Recently mice ILCs but was not required for their development (Rankin et al., lacking AhR have been shown to exhibit a significant deficit in 2013). In contrast to NK cells, we did not observe any expression + CD4− LTi cells and NKp46+ ILCs in the intestinal mucosa (Kiss of Eomes in NKp46 ILCs or LTi cells indicating that Eomes and et al., 2011; Qiu et al., 2012). As the RorγtILCswerenotcom- T-bet operate in a non-redundant manner in this lineage. This + pletely ablated, it appears that AhR is required for the expansion identifies T-bet as a master regulator of NKp46 ILC differenti- − and survival of CD4− LTi cells and NKp46+ ILCs following micro- ation and the CD4 LTi subset as their direct precursor through bial colonization of the intestine after birth rather than their the T-bet-Notch signaling pathway (Rankin et al., 2013). development. CD4+ LTi cells were also affected albeit mildly (Kiss et al., 2011). Extending these studies in an elegant set of CONCLUSION experiments it was shown that although AhR-deficient mice lack In the past 3 years, it has been clear that innate lymphocytes ILFs and CPs, they have normal Peyer’s patch development (Lee comprise a heterogeneous group of cells composed of individ- et al., 2011). Thus, AhR is specifically required in the forma- ual populations with discrete, yet complementary functions in tion of postnatal lymphoid tissues highlighting the differential maintaining tissue homeostasis and providing protection during requirements of Rorγt+ ILC subsets for ILF and Peyer’s patch an immune responses. development. Investigation into AhR target genes required for Teasing apart the intrinsic and extrinsic regulatory circuits ILC development showed that inhibition of the Notch signal- that result in the development and functional contributions of ing pathway, which is regulated by AhR, critically affected ILC the various innate lymphocyte populations during inflammation populations. or infection is both an exciting and evolving story. Significant

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progress has been made in identifying some of the key transcrip- family. In addition, the degree of plasticity between the vari- tional regulators required for the differentiation of ILC subsets, ous Rorγt+ populations is not yet clear. It will be important but how they are regulated remains unclear. For example, whether to uncover whether NKp46+ ILCs are a terminally differenti- Id2 lies up- or downstream of Nfil3 will require re-examination; ated cell type or, as shown for Th cell subsets, they are in or where the terminal differentiation of Rorγt-dependent lineages dynamic equilibrium with CD4− LTi cells responding to environ- lies, especially where LTi cells branch off from NCR+ and NCR− mental cues. ILC3s. This may also facilitate further understanding of whether A third key question for the field revolves around determining different phenotypes of cells within an ILC population represents the identity of the CILP as well as the molecules and steps involved a unified lineage that differs because of localization in the body, in lineage branching to form the different ILC subsets. Id2 is an or they are related but distinct populations within a lineage (e.g., essential core regulator of the development of the innate lympho- understanding the relationship between the highly similar nuo- cyte family and although the exact identity of the CILP remains to cytes and natural helper cells). Future studies will be important in be elucidated it will most likely be an Id2 expressing cell. dissecting these pathways and in understanding the specific contri- Finally, although it is widely recognized that Id2 and Rorγt butions ILC subsets make to immunity and pathology in different are essential regulators of a number of ILC populations, the inflammatory settings. downstream molecular targets of these transcription factors in In the adult tissues, particularly the gut, how the different sub- the LNs and Peyer’s patch have not yet been identified. At a sets of Rorγt+ ILCs are developmentally related has remained cellular level, elucidating whether the attributes of different ILC contentious. It is possible that NKp46+ ILCs develop as direct populations are shared or distinct, whether there exists plasticity progeny of LTi cells or alternately they may arise from a closely between the populations and whether they develop locally from related but distinct cell type. Our recent data show that NKp46+ precursors, or alternately are recruited through the blood will be ILCs are in fact the direct progeny of CD4− LTi cells isolated important in understanding how ILCs orchestrate robust mucosal from the small intestinal lamina propria. However, in a separate protection and provide insight into potential avenues to harness study, LTi cells cultured in vitro under various conditions were and manipulate these cells to promote or ameliorate immune unable to be induced to express NKp46 supporting that the adult responses. phenotypes of gut ILCs remain stable (Sawa et al., 2011). This may be because the conditions used in these experiments were ACKNOWLEDGMENTS not optimal to induce differentiation. Nevertheless, a highly spe- This work is supported by the National Health and Medical cific set of signals from the environment and neighboring cells Research Council (NHMRC) of Australia. Gabrielle T. Belz is that induce transcription factors such as T-bet, Notch, Ahr, and supported by an Australian Research Council Future Fellowship, other signaling molecules drive the differentiation of NKp46+ Lucille Rankin is supported by an NHMRC Dora Lush Fellowship ILCs. However, in order to understand how innate lymphocytes and Joanna Groom is supported by an NHMRC International are regulated in the gut during steady state and disease to effect Postdoctoral Fellowship. This work was made possible through protective immunity, it will be essential to elucidate further the Victorian State Government Operational Infrastructure Support developmental and micro-environmental requirements of the ILC and Australian Government NHMRC IRIIS.

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a T(H)17 cytokine, mediates Conflict of Interest Statement: The Citation: Rankin L, Groom J, Mielke LA, Copyright © 2013 Rankin, Groom, IL-23-induced dermal inflamma- authors declare that the research was Seillet C and Belz GT (2013) Diver- Mielke, Seillet and Belz. This is an tion and acanthosis. Nature 445, conducted in the absence of any com- sity, function, and transcriptional regu- open-access article distributed under 648–651. mercial or financial relationships that lation of gut innate lymphocytes. Front. the terms of the Creative Commons Zheng, Y., Valdez, P. A., Danilenko, D. could be construed as a potential con- Immunol. 4:22. doi: 10.3389/fimmu. Attribution License, which permits use, M., Hu, Y., Sa, S. M., Gong, Q., flict of interest. 2013.00022 distribution and reproduction in other et al. (2008). Interleukin-22 mediates forums, provided the original authors and early host defense against attaching Received: 04 November 2012; accepted: This article was submitted to Frontiers in source are credited and subject to any and effacing bacterial pathogens. Nat. 16 January 2013; published online: 04 NK Cell Biology, a specialty of Frontiers copyright notices concerning any third- Med. 14, 282–289. March 2013. in Immunology. party graphics etc.

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