Accepted Manuscript Title: Human innate lymphoid cells Author: Elisa Montaldo Paola Vacca Chiara Vitale Francesca Moretta Franco Locatelli Maria Cristina Mingari Lorenzo Moretta PII: DOI: Reference:

S0165-2478(16)30007-4 http://dx.doi.org/doi:10.1016/j.imlet.2016.01.007 IMLET 5818

To appear in:

Immunology Letters

Received date: Accepted date:

11-1-2016 25-1-2016

Please cite this article as: Montaldo Elisa, Vacca Paola, Vitale Chiara, Moretta Francesca, Locatelli Franco, Mingari Maria Cristina, Moretta Lorenzo.Human innate lymphoid cells.Immunology Letters http://dx.doi.org/10.1016/j.imlet.2016.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Human innate lymphoid cells Elisa Montaldoa, Paola Vaccab, Chiara Vitaleb,c, Francesca Morettad,e, Franco Locatellif,g, Maria Cristina Mingarib,c*, Lorenzo Morettah. a

U.O.C. Clinical and Experimental Immunology, IRCCS Giannina Gaslini Institute, Genova, Italy

b

Department of Experimental Medicine, University of Genova, Genova, Italy;

c

U.O. Immunology IRCCS AOU San Martino-IST, Genova, Italy;

d

Department of Medicine, University of Verona, Verona, Italy;

e

Ospedale Sacro Cuore Negrar, Verona, Italy;

f

Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesù Children's Hospital,

Rome, Italy; g

Department of Pediatrics, University of Pavia, Pavia, Italy;

h

Department of Immunology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy

*Corresponding Author: Maria Cristina Mingari Address: U.O. Immunologia Pad90 – IRCCS AOU San Martino-IST Largo R. Benzi 10 16132 Genova, Italy email: [email protected] Phone: +39-010-5558210 Fax: +39-010-354282

Highlights 

ILC1, ILC2, and ILC3 have been identified in humans



The NK- and ILC3-committed precursors have been identified



Eomes+ NK cell differentiation is supported by IL-15



RORγt+ ILC3 differentiation is supported by SCF, IL-7, and AhR

1

Abstract The interest in innate lymphoid cells (ILC) has rapidly grown during the last decade. ILC include distinct cell types that are collectively involved in host protection against pathogens and tumor cells and in the regulation of tissue homeostasis. Studies in mice enabled a broad characterization of ILC function and of their developmental requirements. In humans all mature ILC subsets have been characterized and their role in the pathogenesis of certain disease is emerging. Nonetheless, still limited information is available on human ILC development. Indeed, only the cell precursors committed towards NK cells or ILC3 have been described. Here, we review the most recent finding on human mature ILC, discussing their tissue localization and function. Moreover, we summarize the available data regarding human ILC development.

Keywords: ILC, ILC development, NK cells, NK cell differentiation, HSCT

1. Introduction Innate lymphoid cell (ILC) are a heterogeneous population of cells that have been subject of intense research during the past few years. ILC are innate lymphocytes that, unlike adaptive T and B lymphocytes do no express rearranged antigen specific receptors[1]. ILC effector function and transcription factor requirement partially resemble those of T lymphocytes[2]. Accordingly, ILC have been classified into killer-ILC and helper-ILC that mirror CD8+ cytotoxic T cells and CD4+ T helper cells, respectively[1]. Killer-ILC are represented by natural killer (NK) cells, the first ILC subset identified already in the 1970s[3]. NK cells display cytolytic activity and produce cytokines, primarily IFNγ, and mediate host defences against both tumor and virus-infected cells. NK cells express Eomesodermin (Eomes) and T-box transcription factor T-bet, required for their development and function. Helper-ILC are further classified into ILC1, ILC2, and ILC3. ILC1 express T-bet and secrete IFNγ, but different from NK cells, do not exert cytolytic activity. ILC1 have been shown to be involved in responses against protozoa and intracellular bacteria. ILC2 depend, for their development, on expression of GATA binding protein 3 (GATA3) and produce primarily IL-13 and IL-5. They contribute to the defence against helminthes and are involved in allergic responses. Finally, ILC3 express the retinoic acid receptor related orphan receptor (RORγt) and produce “type-17” cytokines, mainly IL-17 and IL-22. ILC3 include fetal lymphoid inducer (LTi) cells, which drive secondary lymphoid organ development during embryogenesis, and postnatal ILC3 that are involved in tissue homeostasis and defence against extracellular pathogens. The majority of studies that allowed the characterization of ILC function and development have been

2

performed in mice[1,4,5]. Here we will review our current knowledge on ILC tissue distribution, function and development in humans.

2. Human ILC localization and function 2.1 NK cells vs ILC1 NK cells were the first ILC subset to be identified. Accordingly, they are the most widely characterized ILC population [6]. While helper-ILC are scarcely represented in peripheral blood (PB), NK cells may represent up to 15% of peripheral blood (PB) lymphocytes. NK cells include two main subsets, i.e. CD56brightCD16- cells and CD56dimCD16+ cells, that differ in terms of phenotype, effector function, and tissue localization. CD56dim cells account for ≈ 90% of PB NK cells. CD56dim NK cells express the Fcγ receptor CD16, through which they can exert the antibody dependent cell-mediated cytotoxicity (ADCC). Moreover, NK cells mediate “natural cytotoxicity” via a set of activating receptors, which recognize their ligands on tumor or virus-infected cells. The main activating NK receptors are the natural cytotoxicity receptors (NCR, i.e. NKp46, NKp30 and NKp44), NKG2D, and DNAM-1[6]. NK cells also express HLA-class I specific inhibitory receptors that prevent killing of autologous normal cells. In particular, CD56dim NK cells express Killer Immunoglobulin Receptors (KIRs) and CD94/NKG2A[6]. CD56dim NK cells express high levels of perforine and granzyme that mediate high cytotoxic activity. Moreover, CD56dim cells produce cytokines in response to the engagement of activating receptors. On the contrary, CD56bright(CD16-KIR-NKG2A/CD94+perforinelow) cells are poorly cytotoxic and are major cytokine producer in response to cytokines, such as IL-12, IL-18, or IL-15. While CD56bright NK cells constitute the minority of PB NK cells, they represent the large majority of NK cells in secondary lymphoid organs. Of note, CD56dim and CD56bright differ in the expression of chemokine receptors, and their tissue distribution in healthy tissues is in accordance with the pattern of expression of chemotactic factor in solid organs. However, most of NK cells present in normal tissues are CD56bright[7]. Recent evidences in mice and humans have suggested that ILC1 represent a resident population within tissues while being barely detectable in PB. ILC1 differ from NK cells because they lack the expression of Eomes and do not exert perforin-dependent cytolytic activity. Studies in mice suggested that Tbet+Eomes+ NK cells and Tbet+Eomes- ILC1 develop from distinct precursor cells of bone marrow and peripheral origin, respectively[8]. However, several phenotypic overlaps exist between NK cells and ILC1[9-11]. Human CD127+Tbet+Eomes-IFNγ+(CD161+CD56-NKp44-KIRPerforine-) ILC1 have been identified in the gut and have been shown to be enriched in the intestine of Crohn disease patients[12]. Concomitantly, Fuchs et al. defined “intraepithelial ILC1” a 3

population of CD127+ in the gastrointestinal epithelia and in tonsils, which was characterized by the CD56+NKp44+CD103+

phenotype,

but

also

expressed

Eomes.

Subsequently,

CD127+CD161+NKp44- ILC1 have been identified also in the skin [13]. Interestingly, Marquardt et al. identified a population of intrahepatic CD3-CD56+CD49a+(NKp44-CD103-KIR+CD57-) that expressed Tbet, but not Eomes, similar to murine liver ILC1[14]. The presence of CD127+Tbet+Eomes-IFNγ+ ILC1 has also been reported in human decidua[15], however, Eomes+ NK cells represent the large majority of lymphocytes in this tissue[11]. Of note, most human decidual and endometrial Eomes+ NK cells express CD49a, as liver ILC1[14]. Moreover, they can express CD103 and CD9 (markers of TGFb-exposure) and NKp44 similarly to the “intraepithelial ILC1”[10]. Thus, in humans, a clear distinction between ILC1 and NK cells is not easy. Tissue specific factors may influence the features of resident ILC populations and/or drive phenotypic changes in NK cells migrated from PB. An extensive transcriptional analysis would help defining whether ILC1 and NK cells indeed represent distinct developmental lineages as reported in mice.

2.2 ILC2 Group 2 ILC were initially identified in mice as an innate source of type-2 cytokines in 2001[16], but they were fully characterized only in 2010[17-19]. Later, Spits and co-workers identified also humans ILC2 in fetal and adult gut and lung and in PB[20]. These cells are also present in the skin[21,22], in the adipose tissue[23], and in tonsils[12]. Experimental murine models have demonstrated that ILC2 contribute to anti-helminthic responses. However, they may also play a pathogenic role in experimental models of asthma, and of lung and skin diseases[24]. Although alterations of the ILC2 proportion have been reported in PB of patients experiencing helminthic infections, the actual involvement of these cells in human anti-helminthic responses has yet to be defined[25]. Of note, ILC2 accumulate in the skin of patients with atopic dermatitis (AD)[26,27] and in the nasal polyps of patients with chronic rhino sinusitis[20]. Similar to the murine counterpart, human ILC2 express GATA3 transcription factor. Moreover, they express CD161, CD127, CD25 (IL-2Rα), ST2 (IL-33R), and IL-17RA. ILC2 respond to IL-33 and IL-25 (IL-17E) by producing primarily the type-2 cytokines IL-13, IL-5, and IL-4. Nonetheless, ILC2 have been reported to produce also pro-inflammatory cytokines such as GM-CSF and IL-8 as well as IL-3, IL9, and IL-21. ILC2 express the prostaglandin D2 (PGD2) receptor CRTH2, but its expression could be modulated by the tissue specific cell localization[28]. Of note, PGD2 can induce the migration of PB and skin ILC2 as well as enhance their cytokine production[29]. All these finding suggest that human ILC2 may respond to epithelial derived cytokines and to mast cell derived metabolites. In turn, they would contribute to early type-2 responses trough the production of cytokines that can 4

promote eosinophil recruitment and survival, and Th2 polarization. A recent report has also shown that ex vivo isolated and in vitro activated ILC2 express the NKp30 NCR. Moreover, B7-H6mediated cell stimulation via NKp30 induced the production of type-2 cytokines[30]. Notably, B7H6 has a widespread expression in the skin of patients with AD as compared to healthy subjects. This suggests that, in the skin of AD patients, ILC2 may be activated also trough NKp30.

2.3 ILC3 Both murine and human ILC3 are identified as Lin-CD127+RORγt+ cells. ILC3 include two main cell subsets, i.e. fetal LTi and postnatal ILC3. In mice it has been shown the fetal LTi cells are among the first cells to populate the site of lymph node (LN) development. LTi can induce the upregulation of adhesion molecules on stromal cells and the release of chemokines that promote the recruitment of T, B and dendritic cells that would colonize the LN, thanks to the interaction between lymphotoxin αβ and its receptor [31,32]. In humans, lin-CD45+CD127+ LTi cells have been identified in fetal mesenteric LN and spleen during the first and second trimester of pregnancy[33,34].

Similar to murine LTi, they express RORC, LTA, LTB, IL-17, and IL-22,

however, they lack CD4 and express CD161 and CD7. Human LTi cells co-cultured in vitro with stromal cells can induce the up-regulation of ICAM and VCAM[33], suggesting that they may play in vivo a lymphoid tissue inducing activity, similar to murine LTi cells. After birth, murine ILC3 are required for the development of cryptopatches and isolated lymphoid follicles in the gut[35,36], in the remodelling/repair of LN after infection[37], and in the homeostasis of the intestinal epithelial barrier[38-40]. ILC3-derived IL-22 induces the release of antimicrobial proteins by intestinal epithelial cells, thus contributing to host protection against extracellular pathogens[41,42]. In humans, ILC3 have been identified in several organs. Human adult ILC3 were first described in intestine and tonsils as Lin-CD56+ cells characterized by the expression of the typical NK cell receptor NKp44 and by IL-22 production and they were initially termed NK22[43]. According to the proposed classification of adult ILC3 into NCR+ and NCR-, NKp44+ ILC3 were thus re-termed NCR+ILC3[44], however subsets of NKp44- ILC3 can express NKp46[45]. Therefore, the definition of NCR+ILC3 should not only rely on NKp44, but also on NKp46 expression. The different subsets of adult ILC3 differ in their cytokine profile. Besides IL22, tonsil NCR+ILC3 can also secrete IL-2, TNF, GM-CSF, and LIF, depending on distinct stimuli [45,46]. Subsequently, ILC3 have been identified after birth in other organs, including spleen, endometrium, decidua, skin, and lung[11,13,15,21,22,47-49]. Splenic ILC3 are mainly localized in the marginal zone (MZ) and through the expression of BAFF and CD40L they can promote the survival and differentiation of MZ B lymphocytes. Moreover, GM-CSF derived from ILC3 5

promotes the survival and activation of splenic neutrophils, which, in turn, express APRIL that promotes IgA production by MZ B cells[47]. Human decidua contains both NCR+ and NCR- ILC3. As shown for tonsils, the two subsets produce IL-22 and IL-17 and TNF respectively. Notably, CD56+NCR+ ILC3 produce large amounts of IL-8, a key cytokine required during early pregnancy[15]. Both subsets have been shown to promote the up-regulation of adhesion molecules on decidual stromal cells, which might suggest a role of these cells in tissue remodelling. Moreover, decidual NCR+ ILC3, thanks to the production of IL-8 and GM-CSF, promote neutrophil recruitment and survival[50]. More recently, ILC3 have also been identified in human endometrium [11,48]. The presence of ILC3 in skin seems to be associated with inflammatory conditions such as psoriasis[13,21,22]. ILC3 have also been identified in association with non-small cell lung cancer (NSCLC). Interestingly, tumor tissue contained higher proportion of ILC3 than matched normal tissues. Moreover, the ILC3 presence correlated with the density of tertiary lymphoid structures[49].

3. ILC development in humans Studies in mice, that can take advantage of fate mapping or knocking out of selected genes, allowed the elucidation of murine ILC developmental relationships. In particular, analysis of the origin of murine ILC allowed the identification of transcription factors, cytokine and environmental signals driving the differentiation of distinct subsets. The characterization of the ILC developmental hierarchy indicate that differentiation proceeds from the common lymphoid progenitor (CLP), then diverge into NK precursor and common helper-ILC precursor and terminates in distinct mature ILC subsets[1,32]. In humans, it has been shown that CD34+ haematopoietic precursor cells (HPC) isolated from several tissues can give rise to one or more ILC subset[51-56]. However, the human ILC differentiation tree is not yet completely determined (Figure 1). NK cells differentiation has been largely studied and some intermediates of differentiation have been identified, from CD34+ cells to CD56dim NK cells. In the last years, different research groups focused their studies also on human helper-ILC development. Here we will focus on NK cell and ILC3 differentiation. Indeed, recent discoveries on ILC3 lead to a re-evaluation of human NK cell developmental pathway.

3.1 Focus on NK cell and ILC3 development The NK-cell developmental potential have been demonstrated for haematopoietic precursors isolated from several tissues. These include not only primary and secondary lymphoid organs, but also PB, umbilical cord blood (UCB), decidua, liver, and intestine[57-64]. Studies performed starting from the early 1990s to the present suggest that human NK cell differentiation proceeds 6

from a CD34+ HPC to mature NK cells through a CLP and an NK-restricted committed progenitor. In the attempt to identify the human CLP, several cell subsets containing NK cell precursors were identified. In particular, it has been shown that bone marrow (BM) Lin-CD34+CD38+CD10+ cells[63] and UCB Lin-CD34+CD38-CD7+ cells[64] can give rise to T, B, and NK cells. However, later it was shown that UCB CD34+CD45RA+CD7+ cells and UCB and BM CD34+CD38CD45RA+CD10+CD7- cells are not strictly restricted towards lymphoid cells, since they can generate dendritic cells, monocytes, and macrophages[65,66]. A turning point in the analysis of NK cell

development

came

in

2005,

when

Freud

and

colleagues

showed

that

also

CD34+CD45RA+β7+CD10+ cells isolated from secondary lymphoid organs differentiated towards NK cells[57]. Moreover, they identified in tonsils and lymph nodes four discrete stages of NK development according to CD34, CD117, and CD94 expression, and suggested a restriction of developmental fate from a multi-lineage precursor (stage-1) to a mature CD34-CD117CD94+CD56bright

NK

cells

(stage-4)[67].

Indeed,

CD34+CD117-CD94-

stage-1

and

CD34+CD117+CD94- stage-2 NK cells displayed also T and DC differentiation potential, lost by CD34-CD117+CD94- stage-3 NK cells. Of note, although stage-2 and -3 NK cells differentiated into Lin-CD56+ cells, only a minor fraction acquired CD94 expression and they did not acquire cytolytic activity or IFN-γ production[67]. Moreover, when stage-3 NK cells derived in vitro from HSC were sorted and cultured again in NK differentiation media, they only partially differentiated towards CD94+IFN-γ+ cells, suggesting an heterogeneity within this cell subset[52,68]. In light of human ILC3 characterization, the lack of final phenotypic and functional NK maturation of stage-2 and -3 cells can be explained by the substantial overlap with the ILC3 committed progenitor and with the mature ILC3, respectively. Indeed, most “stage-3” NK cells are CD127+RORγt+ and produce IL-22 and IL-8, thus representing ILC3[68-70]. Of note, it has been shown that a subset of tonsil and intestinal lamina propria CD34+ cells express the ILC3 master transcription factor RORγt[54]. CD34+RORγt+ cells express CD117, α4β7, CD45RA, CD161 and, upon stimulation, IL-17A and IL-17F. CD34+RORγt+ cells share their transcriptome signature with mature ILC3, but not with mature NK cells. Moreover, upon culture they generated exclusively NKp44+IL-22+ cells and a transient population of CD161+NKp44+IL-17A+ cells. Conversely, only rare Eomes+CD94+IFNγ+ NK cells could be detected in cultures starting from CD34+RORγt+ cells. Thus, in view of these results, tonsil CD34+CD117+ should no more be considered stage-2 NK cells, but rather ILC3 committed progenitors. Interestingly, CD34+CD117+ cells from other compartments do not express RORγt[54]. Tonsil and intestinal lamina propria are enriched in mature ILC3, which are almost undetectable in PB. Thus, it is conceivable that CD34+ cells might acquire RORγt

7

expression, and thus ILC3 commitment, once migrated to mucosal sites where they would finally differentiate towards ILC3. In view of these findings, the classical stages of NK cell development require a new definition. Notably, a recent study has identified a cell precursor committed towards the NK cell lineage, but not towards helper-ILC[56]. In particular, thanks to the simultaneous analysis of markers previously described

for

the

putative

CLP,

the

authors

identified

a

Lin-CD34+CD38+CD123-

CD45RA+CD7+CD10+CD127- NK-restricted progenitor (NKP) in fetal liver and fetal BM, in UCB, and in adult BM and tonsils. This NKP exclusively differentiates towards Eomes+Tbet+ NK cells either in vitro or when transferred into NOD/SCIDγcnull mice. The NKP-derived NK cells include CD16+ and KIR+ cells, are cytotoxic and produce IFN-γ. Of note, the NKP present in tonsils does not express CD117 and RORγt, thus being distinct from the ILC3 committed progenitor[54,56]. Moreover, besides the identification of the NKP, Renoux and co-workers identified CLP-like cells (Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+CD127+) that could generate NK cells, ILC1, ILC2, ILC3, T and B cells, but not myeloid cells. Further studies are required to elucidate whether, in addition to NKP also a common helper-ILC precursor exists in humans as previously shown in mice.

3.2 Signals regulating NK and ILC3 development Human and murine NK and ILC3 development is guided by related requirements in terms of cytokines and environmental cues. In mice, defects in the signaling of the common cytokine γ-chain receptor negatively affect ILC development[32]. However, distinct ILC subsets depend on distinct γ-chain cytokines. Human NK cell generation relies on IL-2Rβ dependent cytokines. Indeed, already in the 1990s IL-15 or IL-2 were shown to drive the differentiation of precursor cells towards NK cells[71-73]. However, immature precursors can efficiently differentiate into NK cells only in the presence of cytokines, such as Flt3-L and SCF, that could induce generation of a NK committed precursor responsive to IL-15[74,75]. Thus, most of culture media used to study in vitro human NK cell differentiation include Flt3-L, SCF, and IL-7, besides IL-15 (Figure 2). It is now well established that, under these culture conditions, human CD34+ cells can efficiently generate also ILC3 [51,52,54-56]. However, it has been shown that UCB CD34+ cells cultured in the presence of Flt3-L+IL-15 generate mostly NK cells. On the contrary, in the presence of Flt3L+SCF+IL-7 cells were largely enriched in ILC3[52]. Although not requiring Flt3-L, tonsil CD34+RORγt- cells show a similar dependence on IL-15 and IL-7+SCF for their differentiation towards NK cells or ILC3. Committed CD34+RORγt+ cells can generate mature ILC3 in the presence of IL-7 or SCF alone, however, the resulting ILC3 are skewed towards the CD56-NKp448

subset[54]. While human ILC3 development can occur in the absence of IL-15, this cytokine can promote proliferation and differentiation of the ILC3-committed progenitors specifically towards the NKp44+IL-22+ ILC3 subset[54]. Moreover, IL-15 and IL-2 can induce proliferation of tonsil and decidual ILC3 and a partial modulation of the ILC3 phenotype (down-regulation of RORγt) and acquisition of NK cell features (expression of CD94 and IFN-γ), implying a certain degree of plasticity between ILC3 and NK cells[15,46]. The proinflammatory cytokine IL-1β is known to promote cytokine production by mature ILC3[45,46]. IL-1β has also been shown to interfere with the plasticity of mature ILC3 towards NK cells[69]. In contrast, IL-1β has a different effect when acting on immature precursors. Indeed, IL-1β added to culture of UCB CD34+ cells skews differentiation towards NK cells, limiting the ILC3 development[55]. However, the overall effect is an inhibition of the recovery of in vitro differentiated CD56+ ILC (NK cells and ILC3) (Figure 2). Of note, AML-derived IL-1β displays a similar effect on ILC differentiation[76]. Cytokines are not the only players in driving NK cell and ILC3 development. Studies in mice revealed that environmental cues, comprising retinoic acid (RA) and ligands of the aryl hydrocarbon receptor (AhR), are required for efficient ILC3 development[32]. While the role of RA in human ILC development is yet to be defined, evidences exist for a role of AhR in the regulation of ILC3 differentiation. AhR is a ligand-dependent transcription factor expressed by ILC3 in both human and mice[32]. Its ligands are exogenous molecules, such as dioxin, bacterial metabolites, and phytochemicals, and endogenous molecules such as tryptophan metabolites[77]. While fetal murine ILC3 can develop independently of AhR, generation of post-natal ILC3 is impaired in the absence of AhR[35,36,78]. In humans, the development of tonsil CD34+RORγt- cells towards ILC3 is affected by AhR inhibition[54]. Of note, the CD117 expression is reduced in the presence of AhR inhibitor, suggesting that, similar to mice, humans AhR regulates the response to SCF. Notably, removal of SCF and blocking of AhR signaling induces the maximal inhibition on ILC3 development favouring NK cell generation. On the contrary, ILC3-committed cells are not influenced by AhR inhibition. A recent report has shown that AhR inhibition in cultures starting from UCB, BM, and PB CD34+ cells results in enhanced NK cell differentiation (Figure 2)[79]. Taken together, these studies suggest that, in humans, AhR may play a role in the commitment of early progenitors towards the ILC3 lineage. A debated issue concerning ILC development is that of inter-lineage plasticity. Indeed, when cultured in vitro in the presence of peculiar stimuli ILC3 can acquire phenotypic and functional features of ILC1 and/or NK cells. ILC3 cultured in the presence of IL-2, IL-15, IL-12, and AhR inhibitors down-regulate RORγt and up-regulate Tbet and/or Eomes[12,15,46,80,81]. On the other hand, IL-1β promoted the stability of ILC3 lineage[69]. A recent report showed that combination of 9

IL-1β and IL-23 besides promoting ILC3 stability, could also promote the differentiation of CD127+ ILC1 towards ILC3, a process enhanced by RA[81].

3.3 ILC development and HSCT The analysis of cell reconstitution following HSCT offers the great opportunity to study “in vivo” human haematopoiesis and immune cell differentiation[82]. In the context of ILC development, NK cell reconstitution after transplant has been largely addressed. Following CD34+ cell HSCT, NK cells appear early after transplant. CD56brightNKG2A+CD16-KIR- NK cells are generated already after 2 weeks, while the differentiation of CD56dimNKG2A+/-CD16+KIR+ cells requires up to 6-8 weeks, thus further supporting the notion that CD56dim represent more mature cells[83,84]. Regarding ILC different from NK cells, little is known about their generation after transplant. Munneke et al. reported that an increase in circulating helper-ILC is detectable early after HSCT[85]. In particular, they could detect a transient wave of activated ILC3 in the PB. Moreover, the presence of activated helper-ILC correlated with a reduced incidence of graft versus host disease (GvHD). The authors hypothesised that, similar to mice, IL-22 produced by ILC3 may protect mucosal tissues from therapy-induced damages, and thus from GvHD. However, to confirm this hypothesis the role of ILC in tissues should be addressed. Several reports showed that pre and post HSCT treatments can affect NK cell differentiation [8689]. It is of note that ILC development after HSCT might be influenced by the HSC source used for transplant. Indeed, CD34+ cells isolated from the PB of G-CSF-mobilized donors displayed a reduced ability to differentiate in vitro towards ILC3 and NK cells, as compared to CD34+ cells isolated from BM or UCB. Moreover, G-CSF can modulate ILC differentiation from UCB-derived CD34+ cells (Figure 2) [90]. Whether G-CSF may also affect the ILC reconstitution in vivo remains to be defined. Further studies are clearly required to define helper-ILC development after HSCT. Notably, this information is particularly important in view of the role played by ILC in innate defences in the mucosal tissues, in secondary lymphoid tissue reconstitution, and in the repair/protection of mucosal epithelial cells damage by the conditioning therapy or GvHD.

4. Concluding remarks The past few years have witnessed a broad interest in ILC development and lineage relationships. However, further elucidation of the human ILC differentiation hierarchy is required. Indeed, the existence of a common-helper ILC precursor is yet to be defined. Moreover, although ILC2 could be generated in vitro[53,56], the identification of the ILC2 committed precursor and of the signals driving an efficient ILC2 differentiation is still lacking. Furthermore, important questions regarding 10

the role of ILC in homeostasis and disease are still unanswered. A deeper analysis of ILC function and differentiation would thus offer new clues that could be translated into clinics.

Conflict of interest disclosure The authors declare no conflict of interest.

Acknowledgments This work was supported by AIRC: IG2010 project n.10225 (L.M.), IG2014 project n.15283 (L.M.), and “Special Program Molecular Clinical Oncology 5x1000” project n.9962 (L.M.). Ministero della Salute: RO strategici 8/07 (M.C.M.).

References [1]

Diefenbach A, Colonna M, Koyasu S. Development, differentiation, and diversity of innate lymphoid cells. Immunity 2014;41:354-365.

[2]

Annunziato F, Romagnani C, Romagnani S. The 3 major types of innate and adaptive cellmediated effector immunity. The Journal of allergy and clinical immunology 2014.

[3]

Herberman RB, Nunn ME, Holden HT, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. International journal of cancer Journal international du cancer 1975;16:230-239.

[4]

Cortez VS, Robinette ML, Colonna M. Innate lymphoid cells: new insights into function and development. Current opinion in immunology 2015;32:71-77.

[5]

McKenzie AN, Spits H, Eberl G. Innate lymphoid cells in inflammation and immunity. Immunity 2014;41:366-374.

[6]

Montaldo E, Del Zotto G, Della Chiesa M, Mingari MC, Moretta A, De Maria A, et al. Human NK cell receptors/markers: a tool to analyze NK cell development, subsets and function. Cytometry Part A : the journal of the International Society for Analytical Cytology 2013;83:702-713.

[7]

Carrega P, Bonaccorsi I, Di Carlo E, Morandi B, Paul P, Rizzello V, et al. CD56(bright)perforin(low) noncytotoxic human NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph. J Immunol 2014;192:3805-3815.

11

[8]

Daussy C, Faure F, Mayol K, Viel S, Gasteiger G, Charrier E, et al. T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow. The Journal of experimental medicine 2014;211:563-577.

[9]

Cortez VS, Fuchs A, Cella M, Gilfillan S, Colonna M. Cutting edge: Salivary gland NK cells develop independently of Nfil3 in steady-state. J Immunol 2014;192:4487-4491.

[10]

Fuchs A, Vermi W, Lee JS, Lonardi S, Gilfillan S, Newberry RD, et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-gammaproducing cells. Immunity 2013;38:769-781.

[11]

Montaldo E, Vacca P, Chiossone L, Croxatto D, Loiacono F, Martini S, et al. Unique Eomes+ NK cell subsets are present in uterus and decidua during early pregnancy. Frontiers in immunology 2016;6.

[12]

Bernink JH, Peters CP, Munneke M, te Velde AA, Meijer SL, Weijer K, et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat Immunol 2013;14:221229.

[13]

Villanova F, Flutter B, Tosi I, Grys K, Sreeneebus H, Perera GK, et al. Characterization of innate lymphoid cells in human skin and blood demonstrates increase of NKp44+ ILC3 in psoriasis. The Journal of investigative dermatology 2014;134:984-991.

[14]

Marquardt N, Beziat V, Nystrom S, Hengst J, Ivarsson MA, Kekalainen E, et al. Cutting edge: identification and characterization of human intrahepatic CD49a+ NK cells. J Immunol 2015;194:2467-2471.

[15]

Vacca P, Montaldo E, Croxatto D, Loiacono F, Canegallo F, Venturini PL, et al. Identification of diverse innate lymphoid cells in human decidua. Mucosal immunology 2015;8:254-264.

[16]

Fort MM, Cheung J, Yen D, Li J, Zurawski SM, Lo S, et al. IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo. Immunity 2001;15:985-995.

[17]

Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, Kawamoto H, et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells. Nature 2010;463:540-544.

[18]

Neill DR, Wong SH, Bellosi A, Flynn RJ, Daly M, Langford TK, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010;464:1367-1370.

[19]

Price AE, Liang HE, Sullivan BM, Reinhardt RL, Eisley CJ, Erle DJ, et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proceedings of the National Academy of Sciences of the United States of America 2010;107:11489-11494.

12

[20]

Mjosberg JM, Trifari S, Crellin NK, Peters CP, van Drunen CM, Piet B, et al. Human IL25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol 2011;12:1055-1062.

[21]

Teunissen MB, Munneke JM, Bernink JH, Spuls PI, Res PC, Te Velde A, et al. Composition of innate lymphoid cell subsets in the human skin: enrichment of NCR(+) ILC3 in lesional skin and blood of psoriasis patients. The Journal of investigative dermatology 2014;134:2351-2360.

[22]

Dyring-Andersen B, Geisler C, Agerbeck C, Lauritsen JP, Gudjonsdottir SD, Skov L, et al. Increased number and frequency of group 3 innate lymphoid cells in nonlesional psoriatic skin. The British journal of dermatology 2014;170:609-616.

[23]

Brestoff JR, Kim BS, Saenz SA, Stine RR, Monticelli LA, Sonnenberg GF, et al. Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 2015;519:242-246.

[24]

Doherty TA. At the bench: understanding group 2 innate lymphoid cells in disease. Journal of leukocyte biology 2015;97:455-467.

[25]

Halim TY. Group 2 innate lymphoid cells in disease. International immunology 2015.

[26]

Kim BS, Siracusa MC, Saenz SA, Noti M, Monticelli LA, Sonnenberg GF, et al. TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation. Science translational medicine 2013;5:170ra116.

[27]

Salimi M, Barlow JL, Saunders SP, Xue L, Gutowska-Owsiak D, Wang X, et al. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. The Journal of experimental medicine 2013;210:2939-2950.

[28]

Tait Wojno ED, Monticelli LA, Tran SV, Alenghat T, Osborne LC, Thome JJ, et al. The prostaglandin D2 receptor CRTH2 regulates accumulation of group 2 innate lymphoid cells in the inflamed lung. Mucosal immunology 2015;8:1313-1323.

[29]

Xue L, Salimi M, Panse I, Mjosberg JM, McKenzie AN, Spits H, et al. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells. The Journal of allergy and clinical immunology 2014;133:1184-1194.

[30]

Salimi M, Xue L, Jolin H, Hardman C, Cousins DJ, McKenzie AN, et al. Group 2 Innate Lymphoid Cells Express Functional NKp30 Receptor Inducing Type 2 Cytokine Production. J Immunol 2016;196:45-54.

[31]

Finke D. Fate and function of lymphoid tissue inducer cells. Current opinion in immunology 2005;17:144-150. 13

[32]

Montaldo E, Juelke K, Romagnani C. Group 3 innate lymphoid cells (ILC3s): Origin, differentiation, and plasticity in humans and mice. European journal of immunology 2015;45:2171-2182.

[33]

Cupedo T, Crellin NK, Papazian N, Rombouts EJ, Weijer K, Grogan JL, et al. Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells. Nat Immunol 2009;10:66-74.

[34]

Hoorweg K, Narang P, Li Z, Thuery A, Papazian N, Withers DR, et al. A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells. J Immunol 2015;195:42574263.

[35]

Kiss EA, Vonarbourg C, Kopfmann S, Hobeika E, Finke D, Esser C, et al. Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles. Science 2011;334:1561-1565.

[36]

Lee JS, Cella M, McDonald KG, Garlanda C, Kennedy GD, Nukaya M, et al. AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch. Nat Immunol 2012;13:144-151.

[37]

Scandella E, Bolinger B, Lattmann E, Miller S, Favre S, Littman DR, et al. Restoration of lymphoid organ integrity through the interaction of lymphoid tissue-inducer cells with stroma of the T cell zone. Nat Immunol 2008;9:667-675.

[38]

Satoh-Takayama N, Vosshenrich CAJ, Lesjean-Pottier S, Sawa S, Lochner M, Rattis F, et al. Microbial Flora Drives Interleukin 22 Production in Intestinal NKp46(+) Cells that Provide Innate Mucosal Immune Defense. Immunity 2008;29:958-970.

[39]

Sonnenberg GF, Monticelli LA, Elloso MM, Fouser LA, Artis D. CD4(+) lymphoid tissueinducer cells promote innate immunity in the gut. Immunity 2011;34:122-134.

[40]

Zenewicz LA, Yancopoulos GD, Valenzuela DM, Murphy AJ, Stevens S, Flavell RA. Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease. Immunity 2008;29:947-957.

[41]

Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. The Journal of experimental medicine 2006;203:22712279.

[42]

Zheng Y, Valdez PA, Danilenko DM, Hu Y, Sa SM, Gong Q, et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nature medicine 2008;14:282-289.

14

[43]

Cella M, Fuchs A, Vermi W, Facchetti F, Otero K, Lennerz JKM, et al. A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature 2009;457:722-725.

[44]

Spits H, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, et al. Innate lymphoid cells - a proposal for uniform nomenclature. Nat Rev Immunol 2013;13:145-149.

[45]

Glatzer T, Killig M, Meisig J, Ommert I, Luetke-Eversloh M, Babic M, et al. RORgammat(+) innate lymphoid cells acquire a proinflammatory program upon engagement of the activating receptor NKp44. Immunity 2013;38:1223-1235.

[46]

Cella M, Otero K, Colonna M. Expansion of human NK-22 cells with IL-7, IL-2, and IL1beta reveals intrinsic functional plasticity. Proceedings of the National Academy of Sciences of the United States of America 2010;107:10961-10966.

[47]

Magri G, Miyajima M, Bascones S, Mortha A, Puga I, Cassis L, et al. Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells. Nat Immunol 2014;15:354-364.

[48]

Doisne JM, Balmas E, Boulenouar S, Gaynor LM, Kieckbusch J, Gardner L, et al. Composition, Development, and Function of Uterine Innate Lymphoid Cells. J Immunol 2015;195:3937-3945.

[49]

Carrega P, Loiacono F, Di Carlo E, Scaramuccia A, Mora M, Conte R, et al. NCR(+)ILC3 concentrate in human lung cancer and associate with intratumoral lymphoid structures. Nature communications 2015;6:8280.

[50]

Croxatto D, Micheletti A, Montaldo E, Orecchia P, Loiacono F, Canegallo F, et al. Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua. Mucosal immunology 2016 accepted for publication

[51]

Tang Q, Ahn YO, Southern P, Blazar BR, Miller JS, Verneris MR. Development of IL-22producing NK lineage cells from umbilical cord blood hematopoietic stem cells in the absence of secondary lymphoid tissue. Blood 2011;117:4052-4055.

[52]

Ahn YO, Blazar BR, Miller JS, Verneris MR. Lineage relationships of human interleukin22-producing CD56+ RORgammat+ innate lymphoid cells and conventional natural killer cells. Blood 2013;121:2234-2243.

[53]

Gentek R, Munneke JM, Helbig C, Blom B, Hazenberg MD, Spits H, et al. Modulation of Signal Strength Switches Notch from an Inducer of T Cells to an Inducer of ILC2. Frontiers in immunology 2013;4:334.

15

[54]

Montaldo E, Teixeira-Alves LG, Glatzer T, Durek P, Stervbo U, Hamann W, et al. Human RORgammat(+)CD34(+)

Cells

Are

Lineage-Specified

Progenitors

of

Group

3

RORgammat(+) Innate Lymphoid Cells. Immunity 2014;41:988-1000. [55]

Ambrosini P, Loiacono F, Conte R, Moretta L, Vitale C, Mingari MC. IL-1beta inhibits ILC3while favoring NK-cell maturation of umbilical cord blood CD34 precursors. European journal of immunology 2015.

[56]

Renoux VM, Zriwil A, Peitzsch C, Michaelsson J, Friberg D, Soneji S, et al. Identification of a Human Natural Killer Cell Lineage-Restricted Progenitor in Fetal and Adult Tissues. Immunity 2015;43:394-407.

[57]

Freud AG, Becknell B, Roychowdhury S, Mao HC, Ferketich AK, Nuovo GJ, et al. A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells. Immunity 2005;22:295-304.

[58]

Vacca P, Vitale C, Montaldo E, Conte R, Cantoni C, Fulcheri E, et al. CD34+ hematopoietic precursors are present in human decidua and differentiate into natural killer cells upon interaction with stromal cells. Proceedings of the National Academy of Sciences of the United States of America 2011;108:2402-2407.

[59]

Moroso V, Famili F, Papazian N, Cupedo T, van der Laan LJ, Kazemier G, et al. NK cells can generate from precursors in the adult human liver. European journal of immunology 2011;41:3340-3350.

[60]

Male V, Hughes T, McClory S, Colucci F, Caligiuri MA, Moffett A. Immature NK cells, capable of producing IL-22, are present in human uterine mucosa. J Immunol 2010;185:3913-3918.

[61]

Mingari MC, Poggi A, Biassoni R, Bellomo R, Ciccone E, Pella N, et al. In vitro proliferation and cloning of CD3- CD16+ cells from human thymocyte precursors. The Journal of experimental medicine 1991;174:21-26.

[62]

Poggi A, Sargiacomo M, Biassoni R, Pella N, Sivori S, Revello V, et al. Extrathymic differentiation of T lymphocytes and natural killer cells from human embryonic liver precursors. Proceedings of the National Academy of Sciences of the United States of America 1993;90:4465-4469.

[63]

Galy A, Travis M, Cen D, Chen B. Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity 1995;3:459-473.

[64]

Hoebeke I, De Smedt M, Stolz F, Pike-Overzet K, Staal FJ, Plum J, et al. T-, B- and NKlymphoid, but not myeloid cells arise from human CD34(+)CD38(-)CD7(+) common lymphoid progenitors expressing lymphoid-specific genes. Leukemia 2007;21:311-319. 16

[65]

Canque B, Camus S, Dalloul A, Kahn E, Yagello M, Dezutter-Dambuyant C, et al. Characterization

of

dendritic

cell

differentiation

pathways

from

cord

blood

CD34(+)CD7(+)CD45RA(+) hematopoietic progenitor cells. Blood 2000;96:3748-3756. [66]

Doulatov S, Notta F, Eppert K, Nguyen LT, Ohashi PS, Dick JE. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat Immunol 2010;11:585-593.

[67]

Freud AG, Yokohama A, Becknell B, Lee MT, Mao HC, Ferketich AK, et al. Evidence for discrete stages of human natural killer cell differentiation in vivo. The Journal of experimental medicine 2006;203:1033-1043.

[68]

Montaldo E, Vitale C, Cottalasso F, Conte R, Glatzer T, Ambrosini P, et al. Human NK cells at early stages of differentiation produce CXCL8 and express CD161 molecule that functions as an activating receptor. Blood 2012;119:3987-3996.

[69]

Hughes T, Becknell B, Freud AG, McClory S, Briercheck E, Yu J, et al. Interleukin-1beta selectively expands and sustains interleukin-22+ immature human natural killer cells in secondary lymphoid tissue. Immunity 2010;32:803-814.

[70]

Hughes T, Becknell B, McClory S, Briercheck E, Freud AG, Zhang X, et al. Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH 17 cytokine interleukin-22. Blood 2009;113:4008-4010.

[71]

Mingari MC, Vitale C, Cantoni C, Bellomo R, Ponte M, Schiavetti F, et al. Interleukin-15induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor. European journal of immunology 1997;27:1374-1380.

[72]

Miller JS, Alley KA, McGlave P. Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor. Blood 1994;83:2594-2601.

[73]

Jaleco AC, Blom B, Res P, Weijer K, Lanier LL, Phillips JH, et al. Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells. J Immunol 1997;159:694-702.

[74]

Mrozek E, Anderson P, Caligiuri MA. Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells. Blood 1996;87:2632-2640.

[75]

Yu H, Fehniger TA, Fuchshuber P, Thiel KS, Vivier E, Carson WE, et al. Flt3 ligand promotes the generation of a distinct CD34(+) human natural killer cell progenitor that responds to interleukin-15. Blood 1998;92:3647-3657. 17

[76]

Vitale C, Ambrosini P, Montaldo E, Ballerini F, Moretta L, Mingari MC. IL-1beta-releasing human acute myeloid leukemia blasts modulate natural killer cell differentiation from CD34+ precursors. Haematologica 2015;100:e42-45.

[77]

Stockinger B, Di Meglio P, Gialitakis M, Duarte JH. The aryl hydrocarbon receptor: multitasking in the immune system. Annual review of immunology 2014;32:403-432.

[78]

Qiu J, Heller JJ, Guo X, Chen ZM, Fish K, Fu YX, et al. The aryl hydrocarbon receptor regulates gut immunity through modulation of innate lymphoid cells. Immunity 2012;36:92104.

[79]

Roeven MW, Thordardottir S, Kohela A, Maas F, Preijers F, Jansen JH, et al. The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34(+) Hematopoietic Stem and Progenitor Cells. Stem cells and development 2015;24:2886-2898.

[80]

Hughes T, Briercheck EL, Freud AG, Trotta R, McClory S, Scoville SD, et al. The transcription Factor AHR prevents the differentiation of a stage 3 innate lymphoid cell subset to natural killer cells. Cell reports 2014;8:150-162.

[81]

Bernink JH, Krabbendam L, Germar K, de Jong E, Gronke K, Kofoed-Nielsen M, et al. Interleukin-12 and -23 Control Plasticity of CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria. Immunity 2015;43:146-160.

[82]

Mackall C, Fry T, Gress R, Peggs K, Storek J, Toubert A. Background to hematopoietic cell transplantation, including post transplant immune recovery. Bone marrow transplantation 2009;44:457-462.

[83]

Moretta L, Montaldo E, Vacca P, Del Zotto G, Moretta F, Merli P, et al. Human natural killer cells: origin, receptors, function, and clinical applications. International archives of allergy and immunology 2014;164:253-264.

[84]

Palmer JM, Rajasekaran K, Thakar MS, Malarkannan S. Clinical relevance of natural killer cells following hematopoietic stem cell transplantation. Journal of Cancer 2013;4:25-35.

[85]

Munneke JM, Bjorklund AT, Mjosberg JM, Garming-Legert K, Bernink JH, Blom B, et al. Activated innate lymphoid cells are associated with a reduced susceptibility to graft-versushost disease. Blood 2014;124:812-821.

[86]

Della Chiesa M, Falco M, Podesta M, Locatelli F, Moretta L, Frassoni F, et al. Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus? Blood 2012;119:399-410.

[87]

Vitale C, Chiossone L, Morreale G, Lanino E, Cottalasso F, Moretti S, et al. Analysis of the activating receptors and cytolytic function of human natural killer cells undergoing in vivo 18

differentiation after allogeneic bone marrow transplantation. European journal of immunology 2004;34:455-460. [88]

Wang H, Grzywacz B, Sukovich D, McCullar V, Cao Q, Lee AB, et al. The unexpected effect of cyclosporin A on CD56+CD16- and CD56+CD16+ natural killer cell subpopulations. Blood 2007;110:1530-1539.

[89]

Locatelli F, Pende D, Mingari MC, Bertaina A, Falco M, Moretta A, et al. Cellular and molecular basis of haploidentical hematopoietic stem cell transplantation in the successful treatment of high-risk leukemias: role of alloreactive NK cells. Frontiers in immunology 2013;4:15.

[90]

Moretta F, Petronelli F, Lucarelli B, Pitisci A, Bertaina A, Locatelli F, et al. The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G-CSF. European journal of immunology 2016.

19

Figure Legends Legend to Figure 1: Model of human ILC development. Based on findings obtained in murine models of ILC development it has been proposed that a similar hierarchal differentiation tree exists in humans. Thus, a hematopoietic stem cell (HSC) would give rise to a common lymphoid progenitor (CLP) capable of giving rise to adaptive and innate lymphocytes. A common ILC precursor (CILP) would then divide in: 1) a branch that differentiate into cytotoxic ILC (i.e. NK cells) trough a NK cell-precursor (NKp); 2) a branch that generate a common helper-ILC precursor (CHILP). The CHILP would further differentiate towards mature helper ILC trough specific ILC precursors (ILC1p, ILC2p, and ILC3p). At present, only the CLP, NKp, and ILC3p have been identified in humans[54,56]. Precursor cells whose existence is yet to be determined are depicted with dashed profile. Transcription factors defining the different mature ILC subsets are indicated in boxes.

20

Legend to Figure 2: Signals driving human NK cell and ILC3 differentiation. Flt3-L, SCF, IL-7, and IL-15 represent the standard cytokine mixture (c-Mix) that drives the differentiation of CD34+ cells towards NK cells and ILC3. However, the selective use of one or more of these cytokines has been shown to preferentially instruct the differentiation towards one cell lineage. Moreover, the presence of additional cytokines or environmental cues can further modulate NK cell and ILC3 development. Distinct culture conditions can also affect the number ofILC generated in culture. Differences in cell number recovery are represented by the size of the arrow.

21