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Understanding human NK cell differentiation: Clues for improving the haploidentical hematopoietic stem cell transplantation
Immunology Letters 155 (2013) 2–5
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Review
Understanding human NK cell differentiation: Clues for improving the haploidentical hematopoietic stem cell transplantation Elisa Montaldo a , Paola Vacca a , Lorenzo Moretta b , Maria Cristina Mingari a,c,∗ a b c
Department of Experimental Medicine, University of Genova, Genova, Italy Giannina Gaslini Institute, Genova, Italy Azienda Ospedaliera Universitaria San Martino–Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
a r t i c l e
i n f o
Article history: Available online 26 September 2013 Keywords: NK cells Allogeneic HSC transplantation NK cell differentiation NK receptors
a b s t r a c t The study of in vitro and in vivo NK cell differentiation from hematopoietic precursors revealed the existence of discrete stages of development. These stages are characterized by the progressive acquisition of markers and receptors that play a crucial role in NK cell function. The knowledge acquired has revealed particularly relevant for improving the HSCT to cure high-risk leukemias in the haplo-HSCT setting, in which NK cells play a central role in the clearance of leukemic cells and in the positive clinical outcome. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Natural killer (NK) cells are important players in innate immunity thanks to their ability to mediate cytolytic activity and to release cytokines on activation [1]. On the basis of the CD56 antigen expression it is possible to distinguish two main human NK cell subsets, referred to as CD56bright and CD56dim cells. CD56bright are predominant in peripheral lymphoid organs and are considered as the cytokine producing subset. In contrast, CD56dim represent the majority of peripheral blood (PB) NK cells and are cytotoxic effector cells [2,3]. Recent studies revealed that “cytotoxic” CD56dim cells can also rapidly release large amounts of cytokines upon receptormediated triggering [4,5]. Thus, two main functional activities of NK cells can be mediated by the same subset within a time period compatible with an innate response. NK cells are finely regulated by a number of receptors with opposite functional capabilities (i.e. inhibitory vs activating). The most relevant inhibitory receptors expressed by NK cells recognize major histocompatibility (MHC) class I molecules. Thanks to the expression of MHC-I molecules, normal cells are protected from the attack of autologous NK cells, whereas transformed or virus-infected cells, which frequently down-regulate MHC-I molecules, become susceptible to NK-mediated killing [6]. In humans, the major inhibitory receptors specific for HLA-I molecules are represented by killer Ig-like receptors (KIRs). They belong to
∗ Corresponding author at: U.O.C. Immunologia-Pad90, AOUSM-IST, Largo R Benzi 10, 16132 Genova, Italy. E-mail address: [email protected] (M.C. Mingari). 0165-2478/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2013.09.022
the immunoglobulin superfamily and are specific for determinants shared by groups of HLA-A, -B or -C alleles. Another important inhibitory receptor is CD94/NKG2A, a heterodimer related to Ctype lectins, specific for the non-classic HLA-class I molecule HLA-E [7–16]. Among the NK activating receptors involved in tumor cell recognition and lysis, the prototypes are collectively referred to as natural cytotoxicity receptors (NCRs), and include NKp46, NKp30 and NKp44 [17–23]. The NCRs ligands are only partially characterized [24–26]. Other activating receptors, that play a relevant role in tumor cell killing are NKG2D, a type II membrane protein characterized by a lectin-like domain that recognizes MICA/B or ULBP proteins [27], and DNAM-1 that is specific for poliovirus receptor (PVR, CD155) and Nectin-2 (CD112) [28]. In addition, human NK cells may express HLA-I-specific activating receptors, including KIR2DS1 and KIR2DS4 and CD94/NKG2C [14,16]. Understanding the mechanism regulating NK cells differentiation from hematopoietic stem cells (HSC) has become an important issue because of the role exerted by NK cells in determining the positive clinical outcome inpatients undergoing allogeneic HSC transplantation (HSCT).
2. Stages of NK cell differentiation The bone marrow (BM) has usually been considered the only site of human NK cell differentiation. However studies in humans and mice provided evidence that NK cell development can occur also in other sites [29–33]. Most of the present knowledge on human NK cell development comes from in vitro studies that tested the ability of either CD34+ HSC or putative downstream NK cell precursors
E. Montaldo et al. / Immunology Letters 155 (2013) 2–5
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Fig. 1. Schematic representation of human NK cell differentiation from CD34+ HSC. Sequential acquisition of markers and functional properties as revealed by in vitro and in vivo studies.
to differentiate towards NK cells upon culture in the presence of appropriate cytokines [34]. These studies suggested that NK cells originate from CD34+ HSCs through discrete stages of development, characterized by the sequential acquisition of receptors and functional capabilities, and that they share developmental relationships with cells of other lineages. Indeed NK cells derive from common lymphoid progenitors (CLP) that can give rise also to T and B lymphocytes [35]. Moreover they share a common precursor with other innate lymphoid cells (ILC) and can also differentiate from myeloid cell progenitors [36–39]. NK cell precursors are characterized by the expression of the inhibitor of DNA binding 2 (ID2) transcription factor (TF) and their progression towards the NK cell lineage is regulated by other TFs such as nuclear factor IL-3 regulated (NFIL3, also termed E4BP4) and thymocyte selection-associated HMG box factor (TOX). The acquisition of mature NK cell function is related to the presence of Eomesodermin (EOMES) and of T-cell-specific T-box transcription factor (Tbet) that induce, respectively, the expression of the cytolytic machinery (i.e. perforin and granzymes) and of IFN␥ [40–42]. In parallel with TFs, surface molecules are gradually acquired during NK cell differentiation. As mentioned above, NK cell precursors derive from CLP and indeed CD34+ CD45RA+ (CD10+ )CD7+ cells isolated from BM are enriched in cells capable of in vitro differentiation towards NK cells [35]. Downstream NK cell precursors, characterized by the peculiar phenotype CD34lo CD45RA+ ˛47hi CD7+/− CD10− , were identified in PB. These cells were also identified in lymph nodes where they are thought to migrate thanks to the expression of ␣47. Freud and Caligiuri provided evidence of the existence, in lymph nodes and tonsils, of different NK cell differentiation stages according to CD34/CD117/CD94/CD56 antigen expression. Indeed they identified: CD34+ CD117− CD94− cells (stage I), CD34+ CD117+ CD94− cells (stage II), CD34− CD117+ CD94− CD56+/− cells (stage III),
CD34− CD117low/− CD94+ CD56bright cells (stage IV) and stage CD34− CD117low/− CD94+/− CD56dim (stage V), and proposed a model of progression from stage I to V [34,43]. The transition from CD56bright to CD56dim is further supported by studies in humans and mice that identified cells with phenotypic and functional properties common to both cell populations [44–46]. Recent evidences would indicate that stage III NK cells may include not only NK cell precursors, but also NCR+ ILC3. Indeed these cells are characterized by a similar phenotype to stage III NK cells (CD34− CD117+ CD94− CD56+/− CD127+ ROR␥t+ NCR+ ), but they also produce IL22 [39,47,48]. Further analysis of human NCR+ ILC3 is needed to clarify whether they represent a distinct cell lineage or an intermediate step of NK cell development. Several studies provided clear evidence that NK cell development may occur in tissues other than BM. Indeed CD34+ HSC and/or NK cell lineage-committed precursors able to give rise to mature NK cells have been isolated from different sites such as PB, umbilical cord blood, thymus, secondary lymphoid organs, fetal or adult liver and decidua. In particular, NK committed CD34+ CD127+ CD122+ (ID2+ E4BP4+ ) cells were identified in decidual tissue [31], CD34+ CD117− CD94− and CD34− CD117+/− CD94−/+ in liver [30] and CD34− CD117+ in intestinal lamina propria [32]. All these immature precursors were shown to undergo differentiation in vitro towards NK cells. These observations support the notion that multiple sites can support NK cell differentiation, and suggested that CD34+ cells or NK-lineage-committed precursors may re-circulate from BM to peripheral tissues where they eventually undergo differentiation towards NK cells. It should be stressed that these studies did not discriminate between NK cells and NCR+ ILC3 and further phenotypic and functional studies are in progress in our lab to define this point. During NK cell development in vitro, it is possible to identify different stages of NK differentiation, which partially overlap with
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E. Montaldo et al. / Immunology Letters 155 (2013) 2–5
those previously described in vivo. In particular, CD34+ cells at early stages acquire CD117 and CD161 that allow the definition of the NK cell precursor (NKP) stage. CD56 molecule is expressed at a subsequent stage. Interestingly, the activating receptor NKp44 is the first NCR to be expressed during early NK cell development. NKp46, NKp30, NKG2D and DNAM-1 activating receptors are acquired at a later stage (Fig. 1). The first inhibitory receptor to be expressed during in vitro (and in vivo) NK cell differentiation is CD94/NKG2A. In particular CD94 acquisition corresponds to CD117 down-regulation, in accordance to ex vivo data [34]. These phenotypic changes shortly precede the expression of the LFA-1 adhesion molecule. When CD56+ cells express LFA-1, they acquire functional features typical of mature NK cells (i.e. cytolytic activity and IFN␥ production) [49,50]. It is of note that precursors with the potential of differentiating towards NK cells may co-express CD33, a marker typical of the myeloid lineage. Indeed, progenitors expressing myeloid markers (CD33/CD115/CD14) have been shown to undergo differentiation towards NK cells in the presence of corticosteroids and/or appropriate cytokines, thus indicating some degree of plasticity between myeloid and NK cell lineages [36–38]. Immature NK cells express surface receptors capable of inducing cell activation, including NKp44 and CD161. Remarkably, CD161 is unable to induce activation in mature NK cells, while its cross-linking induces strong IL-8 production in immature NK cell precursors [49]. On the contrary, 2B4 co-receptor, which functions as an activating receptor in mature NK cells, displays an inhibitory activity in immature NK cells reflecting the lack of expression of the SAP adaptor molecule [51]. Mature NK cells are first characterized by the full expression of activating receptors. Remarkably these cells express as the only HLA-I inhibitory receptor the HLA-E-specific CD94/NKG2A heterodimer. Notably, while the earlier stages of NK cell maturation cannot be readily detected following HSCT, because their differentiation site is mostly the BM, CD56bright CD94/NKG2A+ cells represent the first wave of lymphoid cells detectable in PB 2–3 weeks after HSCT [52]. Further steps of peripheral “mature” NK cell differentiation are marked by the acquisition of potent lytic machinery, thank to the expression of perforin and granzymes. These cells display a lower level of expression of CD56 and acquire KIRs, CD16 and, subsequently, CD57 (considered a marker of terminal NK cell differentiation). CD56dim NK cells not only display a strong cytolytic activity, but also can release abundant cytokines/chemokines (including IFN␥, TNF, CCL3) upon receptor mediated cell triggering [4,5]. CD56dim KIR+ cells can be hardly generated in vitro, whereby IL21 has been shown to facilitate KIR expression [53]. In patients receiving HSCT, CD56dim KIR+ cells are detected 6–8 weeks after transplantation. Remarkably, the appearance of KIR+ cells in the case of haploidentical HSCT coincides with detection of NK alloreactivity. Alloreactive NK cells are characterized by the expression of inhibitory KIRs specific for HLA-I alleles that are not expressed by the recipient as well as by activating KIRs, primarily KIR2DS1 recognizing HLA ligands (C2 in the case of KIR2DS1) present on patient cells [54,55]. The presence and the proportion of alloreactive NK cell populations directly correlate with the clinical outcome in patients with high risk leukemia undergoing aploHSCT, thanks to their ability to kill leukemic cells residual after chemo/radiotherapy. Thus, it is crucial to identify the donor with a “perfect mismatch”, i.e. with high proportions of alloreactive NK cells [52,56,57]. In view of the time interval required for the generation of alloreactive NK cells after HSCT, new protocols have been developed, in which donor derived NK cells are infused together with CD34+ cell precursors. The criteria for donor selection and the clinical aspects of HSCT are described in details in this issue by Locatelli and coworkers.
A recent study has revealed the remarkable effect of CMV infection on the time interval required for NK cell development. Indeed, in patients experiencing CMV reactivation (a frequent event following HSCT), CD56dim KIR+ CD57+ cells appear more rapidly. Thus, CMV infection would favour the generation of alloreactive NK cells. In contrast, NK cell maturation is delayed in CMV uninfected patients [58]. The effect of CMV infection in induction of NK cell maturation and in shaping the NK receptor repertoire is discussed by A. Moretta and co-worker in this issue. 3. Concluding remarks The recent progresses in understanding HSC differentiation, with particular focus on the generation and development of innate lymphoid cells, has offered important clues not only for a better knowledge of the pathophysiology of these cells, but also for improving HSCT, in particular in the haploidentical setting. For example, the study of in vitro NK cell differentiation allowed the definition of different maturational stages characterized by the progressive expression of markers and receptors. In addition, for some of this molecules novel important functional capabilities have been identified (i.e. 2B4 functioning as an inhibitory receptor and CD161 functioning as an activating receptor at the early stages of NK cell differentiation). Moreover, it has been shown that cell precursors expressing typical myeloid markers including CD33, CD115 and CD14 may undergo differentiation towards the NK cell lineage. This occurs under the influence of IL-8 and steroids. These findings have important implications in haploidentical-HSCT in which the generation of large numbers of alloreactive NK cells is crucial for the successful clinical outcome. Remarkably, different from NK cells, limited information is so far available on the in vivo differentiation of other ILC populations. In view of the potential role of these cells in innate, i.e. rapid, defences against different pathogens as well as in the reconstitution of lymphoid tissue, it is important to investigate the development and the role of these cells. Notably, development of T cells displaying similar patterns of cytokine production and defensive capabilities is rather delayed in HSCT. Conflict of interest The authors disclaim no conflict of interest. References [1] Trinchieri G. Biology of natural killer cells. Adv Immunol 1989;47:187–376. [2] Lanier LL, Chang C, Azuma M, Ruitenberg JJ, Hemperly JJ, Phillips JH. Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56). J Immunol 1991;146:4421–6. [3] Caligiuri MA. Human natural killer cells. Blood 2008;112:461–9. [4] De Maria A, Bozzano F, Cantoni C, Moretta L. Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on activation. Proc Natl Acad Sci USA 2011;108:728–32. [5] Fauriat C, Long EO, Ljunggren HG, Bryceson YT. Regulation of human NKcell cytokine and chemokine production by target cell recognition. Blood 2010;115:2167–76. [6] Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2deficient lymphoma variants suggests alternative immune defence strategy. Nature 1986;319:675–8. [7] Moretta A, Bottino C, Pende D, Tripodi G, Tambussi G, Viale O, et al. Identification of four subsets of human CD3− CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition. J Exp Med 1990;172:1589–98. [8] Moretta A, Tambussi G, Bottino C, Tripodi G, Merli A, Ciccone E, et al. A novel surface antigen expressed by a subset of human CD3− CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function. J Exp Med 1990;171:695–714. [9] Moretta A, Vitale M, Bottino C, Orengo AM, Morelli L, Augugliaro R, et al. P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J Exp Med 1993;178:597–604.
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Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Review
Understanding human NK cell differentiation: Clues for improving the haploidentical hematopoietic stem cell transplantation Elisa Montaldo a , Paola Vacca a , Lorenzo Moretta b , Maria Cristina Mingari a,c,∗ a b c
Department of Experimental Medicine, University of Genova, Genova, Italy Giannina Gaslini Institute, Genova, Italy Azienda Ospedaliera Universitaria San Martino–Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
a r t i c l e
i n f o
Article history: Available online 26 September 2013 Keywords: NK cells Allogeneic HSC transplantation NK cell differentiation NK receptors
a b s t r a c t The study of in vitro and in vivo NK cell differentiation from hematopoietic precursors revealed the existence of discrete stages of development. These stages are characterized by the progressive acquisition of markers and receptors that play a crucial role in NK cell function. The knowledge acquired has revealed particularly relevant for improving the HSCT to cure high-risk leukemias in the haplo-HSCT setting, in which NK cells play a central role in the clearance of leukemic cells and in the positive clinical outcome. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Natural killer (NK) cells are important players in innate immunity thanks to their ability to mediate cytolytic activity and to release cytokines on activation [1]. On the basis of the CD56 antigen expression it is possible to distinguish two main human NK cell subsets, referred to as CD56bright and CD56dim cells. CD56bright are predominant in peripheral lymphoid organs and are considered as the cytokine producing subset. In contrast, CD56dim represent the majority of peripheral blood (PB) NK cells and are cytotoxic effector cells [2,3]. Recent studies revealed that “cytotoxic” CD56dim cells can also rapidly release large amounts of cytokines upon receptormediated triggering [4,5]. Thus, two main functional activities of NK cells can be mediated by the same subset within a time period compatible with an innate response. NK cells are finely regulated by a number of receptors with opposite functional capabilities (i.e. inhibitory vs activating). The most relevant inhibitory receptors expressed by NK cells recognize major histocompatibility (MHC) class I molecules. Thanks to the expression of MHC-I molecules, normal cells are protected from the attack of autologous NK cells, whereas transformed or virus-infected cells, which frequently down-regulate MHC-I molecules, become susceptible to NK-mediated killing [6]. In humans, the major inhibitory receptors specific for HLA-I molecules are represented by killer Ig-like receptors (KIRs). They belong to
∗ Corresponding author at: U.O.C. Immunologia-Pad90, AOUSM-IST, Largo R Benzi 10, 16132 Genova, Italy. E-mail address: [email protected] (M.C. Mingari). 0165-2478/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2013.09.022
the immunoglobulin superfamily and are specific for determinants shared by groups of HLA-A, -B or -C alleles. Another important inhibitory receptor is CD94/NKG2A, a heterodimer related to Ctype lectins, specific for the non-classic HLA-class I molecule HLA-E [7–16]. Among the NK activating receptors involved in tumor cell recognition and lysis, the prototypes are collectively referred to as natural cytotoxicity receptors (NCRs), and include NKp46, NKp30 and NKp44 [17–23]. The NCRs ligands are only partially characterized [24–26]. Other activating receptors, that play a relevant role in tumor cell killing are NKG2D, a type II membrane protein characterized by a lectin-like domain that recognizes MICA/B or ULBP proteins [27], and DNAM-1 that is specific for poliovirus receptor (PVR, CD155) and Nectin-2 (CD112) [28]. In addition, human NK cells may express HLA-I-specific activating receptors, including KIR2DS1 and KIR2DS4 and CD94/NKG2C [14,16]. Understanding the mechanism regulating NK cells differentiation from hematopoietic stem cells (HSC) has become an important issue because of the role exerted by NK cells in determining the positive clinical outcome inpatients undergoing allogeneic HSC transplantation (HSCT).
2. Stages of NK cell differentiation The bone marrow (BM) has usually been considered the only site of human NK cell differentiation. However studies in humans and mice provided evidence that NK cell development can occur also in other sites [29–33]. Most of the present knowledge on human NK cell development comes from in vitro studies that tested the ability of either CD34+ HSC or putative downstream NK cell precursors
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Fig. 1. Schematic representation of human NK cell differentiation from CD34+ HSC. Sequential acquisition of markers and functional properties as revealed by in vitro and in vivo studies.
to differentiate towards NK cells upon culture in the presence of appropriate cytokines [34]. These studies suggested that NK cells originate from CD34+ HSCs through discrete stages of development, characterized by the sequential acquisition of receptors and functional capabilities, and that they share developmental relationships with cells of other lineages. Indeed NK cells derive from common lymphoid progenitors (CLP) that can give rise also to T and B lymphocytes [35]. Moreover they share a common precursor with other innate lymphoid cells (ILC) and can also differentiate from myeloid cell progenitors [36–39]. NK cell precursors are characterized by the expression of the inhibitor of DNA binding 2 (ID2) transcription factor (TF) and their progression towards the NK cell lineage is regulated by other TFs such as nuclear factor IL-3 regulated (NFIL3, also termed E4BP4) and thymocyte selection-associated HMG box factor (TOX). The acquisition of mature NK cell function is related to the presence of Eomesodermin (EOMES) and of T-cell-specific T-box transcription factor (Tbet) that induce, respectively, the expression of the cytolytic machinery (i.e. perforin and granzymes) and of IFN␥ [40–42]. In parallel with TFs, surface molecules are gradually acquired during NK cell differentiation. As mentioned above, NK cell precursors derive from CLP and indeed CD34+ CD45RA+ (CD10+ )CD7+ cells isolated from BM are enriched in cells capable of in vitro differentiation towards NK cells [35]. Downstream NK cell precursors, characterized by the peculiar phenotype CD34lo CD45RA+ ˛47hi CD7+/− CD10− , were identified in PB. These cells were also identified in lymph nodes where they are thought to migrate thanks to the expression of ␣47. Freud and Caligiuri provided evidence of the existence, in lymph nodes and tonsils, of different NK cell differentiation stages according to CD34/CD117/CD94/CD56 antigen expression. Indeed they identified: CD34+ CD117− CD94− cells (stage I), CD34+ CD117+ CD94− cells (stage II), CD34− CD117+ CD94− CD56+/− cells (stage III),
CD34− CD117low/− CD94+ CD56bright cells (stage IV) and stage CD34− CD117low/− CD94+/− CD56dim (stage V), and proposed a model of progression from stage I to V [34,43]. The transition from CD56bright to CD56dim is further supported by studies in humans and mice that identified cells with phenotypic and functional properties common to both cell populations [44–46]. Recent evidences would indicate that stage III NK cells may include not only NK cell precursors, but also NCR+ ILC3. Indeed these cells are characterized by a similar phenotype to stage III NK cells (CD34− CD117+ CD94− CD56+/− CD127+ ROR␥t+ NCR+ ), but they also produce IL22 [39,47,48]. Further analysis of human NCR+ ILC3 is needed to clarify whether they represent a distinct cell lineage or an intermediate step of NK cell development. Several studies provided clear evidence that NK cell development may occur in tissues other than BM. Indeed CD34+ HSC and/or NK cell lineage-committed precursors able to give rise to mature NK cells have been isolated from different sites such as PB, umbilical cord blood, thymus, secondary lymphoid organs, fetal or adult liver and decidua. In particular, NK committed CD34+ CD127+ CD122+ (ID2+ E4BP4+ ) cells were identified in decidual tissue [31], CD34+ CD117− CD94− and CD34− CD117+/− CD94−/+ in liver [30] and CD34− CD117+ in intestinal lamina propria [32]. All these immature precursors were shown to undergo differentiation in vitro towards NK cells. These observations support the notion that multiple sites can support NK cell differentiation, and suggested that CD34+ cells or NK-lineage-committed precursors may re-circulate from BM to peripheral tissues where they eventually undergo differentiation towards NK cells. It should be stressed that these studies did not discriminate between NK cells and NCR+ ILC3 and further phenotypic and functional studies are in progress in our lab to define this point. During NK cell development in vitro, it is possible to identify different stages of NK differentiation, which partially overlap with
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those previously described in vivo. In particular, CD34+ cells at early stages acquire CD117 and CD161 that allow the definition of the NK cell precursor (NKP) stage. CD56 molecule is expressed at a subsequent stage. Interestingly, the activating receptor NKp44 is the first NCR to be expressed during early NK cell development. NKp46, NKp30, NKG2D and DNAM-1 activating receptors are acquired at a later stage (Fig. 1). The first inhibitory receptor to be expressed during in vitro (and in vivo) NK cell differentiation is CD94/NKG2A. In particular CD94 acquisition corresponds to CD117 down-regulation, in accordance to ex vivo data [34]. These phenotypic changes shortly precede the expression of the LFA-1 adhesion molecule. When CD56+ cells express LFA-1, they acquire functional features typical of mature NK cells (i.e. cytolytic activity and IFN␥ production) [49,50]. It is of note that precursors with the potential of differentiating towards NK cells may co-express CD33, a marker typical of the myeloid lineage. Indeed, progenitors expressing myeloid markers (CD33/CD115/CD14) have been shown to undergo differentiation towards NK cells in the presence of corticosteroids and/or appropriate cytokines, thus indicating some degree of plasticity between myeloid and NK cell lineages [36–38]. Immature NK cells express surface receptors capable of inducing cell activation, including NKp44 and CD161. Remarkably, CD161 is unable to induce activation in mature NK cells, while its cross-linking induces strong IL-8 production in immature NK cell precursors [49]. On the contrary, 2B4 co-receptor, which functions as an activating receptor in mature NK cells, displays an inhibitory activity in immature NK cells reflecting the lack of expression of the SAP adaptor molecule [51]. Mature NK cells are first characterized by the full expression of activating receptors. Remarkably these cells express as the only HLA-I inhibitory receptor the HLA-E-specific CD94/NKG2A heterodimer. Notably, while the earlier stages of NK cell maturation cannot be readily detected following HSCT, because their differentiation site is mostly the BM, CD56bright CD94/NKG2A+ cells represent the first wave of lymphoid cells detectable in PB 2–3 weeks after HSCT [52]. Further steps of peripheral “mature” NK cell differentiation are marked by the acquisition of potent lytic machinery, thank to the expression of perforin and granzymes. These cells display a lower level of expression of CD56 and acquire KIRs, CD16 and, subsequently, CD57 (considered a marker of terminal NK cell differentiation). CD56dim NK cells not only display a strong cytolytic activity, but also can release abundant cytokines/chemokines (including IFN␥, TNF, CCL3) upon receptor mediated cell triggering [4,5]. CD56dim KIR+ cells can be hardly generated in vitro, whereby IL21 has been shown to facilitate KIR expression [53]. In patients receiving HSCT, CD56dim KIR+ cells are detected 6–8 weeks after transplantation. Remarkably, the appearance of KIR+ cells in the case of haploidentical HSCT coincides with detection of NK alloreactivity. Alloreactive NK cells are characterized by the expression of inhibitory KIRs specific for HLA-I alleles that are not expressed by the recipient as well as by activating KIRs, primarily KIR2DS1 recognizing HLA ligands (C2 in the case of KIR2DS1) present on patient cells [54,55]. The presence and the proportion of alloreactive NK cell populations directly correlate with the clinical outcome in patients with high risk leukemia undergoing aploHSCT, thanks to their ability to kill leukemic cells residual after chemo/radiotherapy. Thus, it is crucial to identify the donor with a “perfect mismatch”, i.e. with high proportions of alloreactive NK cells [52,56,57]. In view of the time interval required for the generation of alloreactive NK cells after HSCT, new protocols have been developed, in which donor derived NK cells are infused together with CD34+ cell precursors. The criteria for donor selection and the clinical aspects of HSCT are described in details in this issue by Locatelli and coworkers.
A recent study has revealed the remarkable effect of CMV infection on the time interval required for NK cell development. Indeed, in patients experiencing CMV reactivation (a frequent event following HSCT), CD56dim KIR+ CD57+ cells appear more rapidly. Thus, CMV infection would favour the generation of alloreactive NK cells. In contrast, NK cell maturation is delayed in CMV uninfected patients [58]. The effect of CMV infection in induction of NK cell maturation and in shaping the NK receptor repertoire is discussed by A. Moretta and co-worker in this issue. 3. Concluding remarks The recent progresses in understanding HSC differentiation, with particular focus on the generation and development of innate lymphoid cells, has offered important clues not only for a better knowledge of the pathophysiology of these cells, but also for improving HSCT, in particular in the haploidentical setting. For example, the study of in vitro NK cell differentiation allowed the definition of different maturational stages characterized by the progressive expression of markers and receptors. In addition, for some of this molecules novel important functional capabilities have been identified (i.e. 2B4 functioning as an inhibitory receptor and CD161 functioning as an activating receptor at the early stages of NK cell differentiation). Moreover, it has been shown that cell precursors expressing typical myeloid markers including CD33, CD115 and CD14 may undergo differentiation towards the NK cell lineage. This occurs under the influence of IL-8 and steroids. These findings have important implications in haploidentical-HSCT in which the generation of large numbers of alloreactive NK cells is crucial for the successful clinical outcome. Remarkably, different from NK cells, limited information is so far available on the in vivo differentiation of other ILC populations. In view of the potential role of these cells in innate, i.e. rapid, defences against different pathogens as well as in the reconstitution of lymphoid tissue, it is important to investigate the development and the role of these cells. Notably, development of T cells displaying similar patterns of cytokine production and defensive capabilities is rather delayed in HSCT. Conflict of interest The authors disclaim no conflict of interest. References [1] Trinchieri G. Biology of natural killer cells. Adv Immunol 1989;47:187–376. [2] Lanier LL, Chang C, Azuma M, Ruitenberg JJ, Hemperly JJ, Phillips JH. Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56). J Immunol 1991;146:4421–6. [3] Caligiuri MA. Human natural killer cells. Blood 2008;112:461–9. [4] De Maria A, Bozzano F, Cantoni C, Moretta L. Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on activation. Proc Natl Acad Sci USA 2011;108:728–32. [5] Fauriat C, Long EO, Ljunggren HG, Bryceson YT. Regulation of human NKcell cytokine and chemokine production by target cell recognition. Blood 2010;115:2167–76. [6] Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2deficient lymphoma variants suggests alternative immune defence strategy. Nature 1986;319:675–8. [7] Moretta A, Bottino C, Pende D, Tripodi G, Tambussi G, Viale O, et al. Identification of four subsets of human CD3− CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition. J Exp Med 1990;172:1589–98. [8] Moretta A, Tambussi G, Bottino C, Tripodi G, Merli A, Ciccone E, et al. A novel surface antigen expressed by a subset of human CD3− CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function. J Exp Med 1990;171:695–714. [9] Moretta A, Vitale M, Bottino C, Orengo AM, Morelli L, Augugliaro R, et al. P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J Exp Med 1993;178:597–604.
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