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Keywords:

  • Natural killer cells precursors;
  • Maturation;
  • Lymphokines;
  • Killer Ig-like receptors

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The NK cell maturation from CD34+ Lin hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect ofdifferent cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34+ Lin cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56+ cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectablein developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56dim cell fraction, while the CD56bright population was only composed of CD94/NKG2A and CD94/NKG2A+ cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.

Abbreviations:
NCR:

Natural cytotoxicity receptors

KIR:

Killer Ig-like receptors

CB:

Cord blood

FL:

Fetal liver tyrosine kinases 3 (Flt3-L)-ligand

SCF:

Stem cell factor

SH2D1A:

Src homology 2 (SH2) domain-containing protein 1A

XLP:

X-linked lymphoproliferative disease

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Human natural killer (NK) cells represent a distinct population of large granular lymphocytes (LGL) that play an important role in the host's early immune response to infection and malignant transformation. The cytolytic function of human NK cells is regulated by two distinct types of receptors: MHC class I-specific receptors, that inhibit cytotoxicity upon recognition of their MHC ligands, and triggering receptors that induce NK-mediated target cell killing as a result of their interaction with non-MHC ligands.

In humans, the inhibitory receptors are represented by the Ig-like killer inhibitory receptors (KIR) specific for HLA-A, -B, or –C, the CD94/NKG2A heterodimer specific for HLA-E, and IL-T2/LIR1, a receptor that displays a broad specificity for different HLA class I molecules 13. The activating receptors are represented by NKp30, NKp44 and NKp46, all confined to NK cells and collectively termed "natural cytotoxicity receptors" (NCR) 4. Another triggering receptor, NKG2D, is expressed by both NK cells and cytolytic T lymphocytes5, 6. Other molecules, mostly functioning as activating coreceptors, are represented by 2B4 molecule that is expressed by NK cells and by a subset of cytolytic Tlymphocytes 7, 8, NTB-A, expressed by all NK, T and B lymphocytes 9, and NKp80, virtually confined to NK cells 10.

Although there have been a number of important advances in our understanding of the NK-mediated target cell recognition, signal transduction and cytokine production, the developmental sequenceleading to mature NK cells is still poorly defined. The process of NK cell differentiation from CD34+ Lin progenitors was thought to be strictly dependent on the direct contact with stromal ligands 11; however, appropriate mixtures of cytokines could obviate the strict requirement for stromal cells 1218.

In a previous study we showed that human NK cell precursors can be derived in vitro by culturing CD34+ Lin cells isolated from umbilical cord blood (CB) in the presence of exogenous cytokines (IL-15, Flt3-L, SCF and IL-7) and stromal cells (MS-5) 19. These culture conditions supported both the proliferation and the maturation of NK cell precursors that progressively acquired various NK cell markers/receptors. Thus, two members of the NCR family, NKp46 and NKp30, were expressed early during maturation and their expression at the cell surface correlated with the acquisition, by immature NK cells, of cytolytic activity against NK-susceptible tumor target cells. Subsequently, developing NK cells expressed CD94/NKG2A, NKG2D and, at least in part, CD16 and NKp80. However, only a small fraction of these cells (2–8%) acquired the ability to express KIR. Moreover, essentially no expression of CD2 and CD8 molecules could be detected. Similar results were reported by other studies thus supporting the notion that only a partial NK cell maturation could be achieved in vitro.

In the present study we show that IL-21 plays a crucial role in inducing an acceleration of the maturation steps towards the acquisition of a mature NK cell phenotype, including the surface expression of KIR, CD2 and CD8.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Phenotypic analysis of CD34+ cells cultured in the presence of IL-21

Human NK cell precursors were derived in vitro, by culturing CD34+ Lin cells isolated from CB in the presence of exogenous cytokines as described 1119. Previous studies indicated that a small fraction (15–20%) of CD56+ NK cell precursors could be detected upon 20–30 days culture of CD34+ Lin cells in the presence of stromal cells (MS5) and cytokines (including IL-15, IL-7, FL and SCF) 19. Similar results could be obtained also by culturing CB-derived CD34+ precursors in the absence of stromal cells although, in this case, NK-cell development was slightly accelerated (S. Sivori, unpublished).

In the present study, CD34+ Lin cells (2×104 cells/well) were cultured in medium containing IL-15, FL and SCF either in the absence or in the presence of recombinant IL-21 20. Proliferating cells were then analyzed, by cytofluorimetric analysis, at various time intervals for the surface expression of different NK cell markers. Cells were first analyzed after 25 days of culture in the presence of IL-21. At this stage, different from previous studies performed in the absence of IL-21, developing NK cells were characterized by a substantial fraction of CD56+ cells (∼50%) that already expressed additional NK cell markers (Fig. 1). These included NKp46 (Fig. 1) and NKp30 (not shown) (in the absence of IL-21, both these receptors were expressed only after 35–40 days), CD94/NKG2A and CD16 (in the absence of IL-21, CD16 is expressed after 45–55 days) (Fig. 1). Thus, both the generation of NK cell precursors (CD56+) and the expression of NK cell maturation markers were remarkably accelerated in the presence of IL-21.

After 35 days of culture, a most remarkable finding was the appearance of a KIR+ NK cell subset (at this stage, cells cultured in the absence of IL-21 were NKG2A, KIR). Moreover, 40–65% of cells expressed CD56, NKp46 and CD94/NKG2A (Fig. 1). Remarkably, the fluorescence intensity of CD56+ precursors was not homogeneous, due to the presence of CD56dim and CD56bright cells. Cells expressing the CD56bright phenotype were characterized by higher levels of surface NKp46 but did not express KIR. On the other hand, the CD56dim cell fraction contained all the KIR+ NK cells and was largely represented by CD16+ cells. CD94/NKG2A was expressed in all CD56dim cells but only in approximately half of the CD56bright subset suggesting that the acquisition of the CD56bright phenotype precedes the surface expression of CD94/NKG2A. This surface phenotype is reminiscent of peripheral blood mature NK cells, in which most KIR+ (and CD16+) cells are confined to the CD56dim cell subset 21 while CD56bright cells express higher surface densities of NCR and NKG2A (S. Sivori, unpublished).

At day 45 of culture in the presence of IL-21, the KIR+ cell fraction was remarkably increased in size reaching, in certain donors, up to ∼60% of the total (CD56+) NK cells (Fig. 1). In this case, most CD56dim cells were KIR+ (30–90%) whereas virtually all CD56bright cells were KIR. Similar results were obtained when CD16+ and CD16 cell fractions were analyzed. In particular, CD16 cells were confined to the CD56bright cell fraction. Although CD94/NKG2A was expressed by the majority of NK cells, a small fraction of CD56bright cells was CD94/NKG2A. As mentioned above, it is possible that this small subset may represent less differentiated NK cells that may differentiate into CD56bright NKG2A+ cells and subsequently into CD56dim KIR+ mature NK cells. Fig. 1 also shows the same CD34+ Lin- population that had been cultured in the absence of IL-21. It is evident the delay in maturation. For example, cells after 45 days of culture had a surface phenotype similar to cells cultured in IL-21 for only 25 days (compare the expression of CD16).

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Figure 1. Progressive acquisition of NK receptors upon culture of CD34+ Lin cell precursors in the presence or in the absence of IL-21. CD34+ Lin CB cells were cultured in the presence of IL-15, Flt3-L, SCF, IL-7 and with or without IL-21 and analyzed by two-color immunofluorescence for the expression of NKp46, NKG2A, KIR and CD16 in combination with CD56 molecules at the indicated time intervals.

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2.2 Analysis of the KIR surface phenotype expressed by NK cell precursors cultured in the presence of IL-21

Fig. 2 shows the analysis of two immature NK cell populations derived from different cord blood samples (CB H7 and CB H18). In both cases KIR+ cells (detected by staining with a mixture of four mAb specific for the most representative KIR) represented appreciable fractions of NK cells, although their percentages displayed major variations in the two populations analyzed (60% and 16%, respectively).

In order to precisely identify the KIR expressed in the presence of IL-21, we further analyzed the reactivity of single anti-KIR mAb. As shown in Fig. 3a, the majority of KIR+ cells, derived from CB H7 at 45 days of culture, expressed KIR2DL1/S1 (identified by the EB6 mAb) while a smaller fraction expressed KIR2DL2/L3/S2 (identified by the GL183 mAb) and/or KIR3DL1/S1 (identified by the Z27 mAb). No cells were stained by the KIR2DS4-specific FS172 mAb (not shown). The KIR phenotype was comparatively analyzed with that of a polyclonal, IL-2-cultured NK cell population derived from CD3 CD56+ mature NK cells isolated from the same cord blood (Fig. 3b). This study revealed that the precursors-derived and the mature NK cell populations had a similar KIR phenotype. On the other hand, differences in the percentage of expression of the various KIR as well as in their reciprocal distribution could be observed. In particular, cells expressing KIR2DL1/S1 represented 29% in the bulk NK cell population and 60% in the precursors-derived NK cells, cells expressing KIR2DL2/L3/S2 were 21 and 6%, respectively while cells expressing KIR3DL1 were 33 and 8%, respectively. Again no KIR2DS4+ cells could be detected. Similar results were obtained by the comparison of mature or precursors-derived NK cell population from CB H18. In this case, most KIR+ cells were stained by GL183 mAb (80%) while cells stained by EB6 or Z27 mAb were 15 and 5%, respectively. Although GL183+ cells were more represented also in mature NK cells, their percentage was lower (45%) than in precursors-derived NK cells (not shown).

The differences in percentages could simply reflect the culture conditions used to obtain the two NK cell populations. Notably, beside the use of different cytokines, a major difference was represented by the allogeneic feeder cells used for growing mature NK cells but not immature NK cell precursors. Indeed, allogeneic feeder cells have previously been shown to have a major impact on the selection of proliferating NK subsets in mature, polyclonal NK populations 1.

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Figure 2. Surface expression of KIR, CD2 and CD8 molecules by NK cell precursors. In these experiments developing NK cells were analyzed after 45 days of culture in the presence of IL-21. Two-color immunofluorescence analysis is reported for the expression of KIR, CD2 and CD8 in combination with CD56.

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Figure 3. KIR expression in immature and mature NK cells. In these experiments we comparatively analyzed the expression of KIR molecules in NK cells derived from CD34+ Lin CB cells cultured in the presence of IL-21 and in IL-2-activated mature NK cells derived from the same cord-blood. Both types of NK cells were stained with a mixture of various anti-KIR mAb and subjected to cytofluorimetric analysis by double fluorescence against CD56 (immature NK cells, panel a) or by one color fluorescence (mature NK cells, panel b).

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2.3 Expression of CD2 and CD8 molecules by NK cell precursors cultured in the presence of IL-21

As shown in previous studies, CD34+ Lin cell precursors cultured either in the absence or in the presence 19 of stromal cells and cytokines (IL-15, FL and SCF and, in some experiments, IL-7) undergo differentiation in NK cells characterized not only by low KIR expression but also by the complete lack of surface CD2 and CD8. Thus, although developing NK cells were characterized by the acquisition of several NK cell markers and receptors, a complete differentiation towards NK cells, with a surface phenotype comparable to that of circulating, mature NK cells, could not be obtained. In the presence of IL-21, both CD2 and CD8 molecules were induced after 35 days of culture in a fraction of NK cell precursors (together with KIR expression). Fig. 2 shows the results of the analysis of two cell populations derived from CB precursors (CB H7 and CB H18) cultured for 45 days in the presence of IL-21. In both instances, CD2, CD8 and KIR were expressed by a subset of NK cells although the percentages of positive cells were different in the two samples analyzed. It is also of note that CD2 and CD8 molecules were not expressed by the same cell population as also suggested by the differences in the percentage of positive cells. Moreover, their expression was not strictly correlated with the KIR expression. Thus, in CB H18 the percentage of CD2+ cells was higher than that of KIR+. Comparable percentages of CD8+ cells were detected in CB H7 and CB H18. It is conceivable, however, that the surface expression of CD2, CD8 and KIR on differentiating NK cells might represent, at least in part, a coordinated process that requires the presence of IL-21. IL-21 may act either directly or indirectly (i.e. by inducing the release of other factors) on developing cells. Finally, in some of the CB-derived precursors analyzed, cells undergoing proliferation were represented not only by NK cell precursors but also by CD3+ T cells. For example, as shown in Fig. 2, CB H18-derived cells contained also a CD2+CD56–/dim population. Further analysis revealed that these cells were characterized by the expression of CD3 with a predominant CD8+ phenotype (not shown).

2.4 Surface expression and function of 2B4 and NTB-A molecules in NK cell precursors cultured in the presence of IL-21

Previous data indicated that the cytolytic activity mediated by NK cell precursors is coincident with the acquisition at the cell surface of triggering receptors such as NKp46 and NKp30. Moreover, immature NK cells have been shown to express surface 2B4 molecules characterized by an inhibitory rather than an activating function 19.

To assess the surface expression and the function of these receptors in IL-21-cultured NK cell precursors, their cytolytic activity was evaluated, at different stages of development, in a redirected killing assay against the FcγR+ P815 murine target cell line. As shown in Fig. 4a, mAb-mediated cross-linking of CD16 and NKp46 induced triggering of cytolytic activity at different stages of development. On the other hand, cross-linking of 2B4 and NTB-A exerted an inhibitory effect in NK cell precursors detectable at 25 days of culture but not at later stages. Thus, after 35 days of culture mAb-mediated cross-linking of both molecules did not modify the levels of cytotoxicity, whereas after 45 days it resulted in an enhancement of target cell lysis.

Differences in the functional capabilities of 2B4 and NTB-A during NK cell maturation in the presence of IL-21 was further confirmed in experiments in which immature NK cells were analyzed for cytotoxicity against the HLA class I and FcγR LCL 721.221 human B cell line.

After 25 days of culture, NK precursors failed to kill this cell line both in the presence and in the absence of IL-21. This effect was due to the inhibitory function of 2B4 and NTB-A, as indicated by the ability to reconstitute lysis upon mAb-mediated masking of these receptors. After 35 days of culture, the spontaneous lysis of the LCL 721.221 cells was slightly increased. At this stage, no reconstitution of cytotoxicity could be obtained by mAb-mediated masking of 2B4 and NTB-A. After 45 days of culture, NK cells display an efficient cytolytic activity. In addition, lysis was partially inhibited by the simultaneous masking of 2B4 and NTB-A. A further inhibition of lysis could be observed by masking NKp46. These data document a progressive shift from the inhibitory to the activating form of 2B4 and NTB-A molecules in NK cells undergoing maturation in the presence of IL-21.

As shown in previous studies, the early stages of NK cell differentiation were characterized by the lack of SH2D1A transcripts 19. Thus, the progressive shift from the inhibitory to the activating form of 2B4 and NTB-A occurring in the presence of IL-21 could reflect the progressive induction of SH2D1A transcripts in NK cell precursors undergoing a more advanced stage of maturation. To verify this hypothesis, we analyzed the presence of SH2D1A-transcripts in NK precursors cultured for various time intervals in the presence of IL-21. As shown in Fig. 4b, reverse transcriptase (RT)-PCR analysis revealed that SH2D1A-transcripts are indeed expressed after 45 days of culture.

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Figure 4. Progressive functional switch of 2B4 and NTB-A molecules during NK cell development. (a) Developing NK cells derived from CD34+ Lin- CB cells cultured in the presence of IL-21 were assessed for cytolytic activity in a redirected killing assay against the FcγR+ P815 target cell line either in the absence or in the presence of the indicated (IgG1) mAb. The E/T ratio was 4:1. The same NK cells were assessed for cytolytic activity in a mAb-mediated masking assay against the FcγR LCL 721.221 cell line either in the presence or in the absence of mAb specific for the indicated molecules. In these experiments of receptor blocking, the mAb used was F(ab′)2 or of IgM isotype. The E/T ratio was 4:1. (b) Expression of SH2D1A transcript in NK cell precursors and mature NK cell populations. RT-PCR products were loaded in a 1.5% agarose gel. Lane 1: NK cell precursors after 25 days of culture; lane 2: NK cell precursors after 45 days of culture; lane 3: mature NK cell population; lane 4: negative control; lane 5: HinfI digested ΦX 174 molecular weight marker. A control amplification with β-actin-specific primers was performed (not shown).

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This study provides evidence that IL-21 plays a crucial role in NK cell development by inducing an accelerated maturation of CD34+ hematopoietic precursors towards cells that display both phenotypic and functional features of fully mature NK cells. Indeed, not only NK cells were generated within a shorter culture period, but they also acquired surface markers, including KIR, CD2and CD8, that in previous studies were either undetectable or expressed by a minor NK cell population. Importantly, in the presence of IL-21 the proportions of KIR+ NK cells approximated that of mature NK cells, reaching up to 60% of the developing NK cells. Thus, the use of IL-21 allows to recapitulate in vitro the whole process of human NK cell maturation and offers an important tool for the analysis of the molecular mechanisms involved in NK cell development and, in particular, in the acquisition of the KIR repertoire.

Most of the models of NK cell differentiation have been based on in vitro culture systems in which NK cells could be generated from CD34+ Lin precursors. These studies helped to identify the soluble growth factors involved in the process of NK cell development 1119.

IL-21 is a recently identified cytokine 20, 22, 23, which is closely related to IL-2 and IL-15. The IL-21-specific receptor (IL-21R) has the highest homology with the IL-2R β-chain and with the IL-4R α-chain. Upon ligand binding, IL-21R associates with the common γ-chain, a property shared with the receptors for IL-2, IL-4,IL-7, IL-9 and IL-15 23. Thus far, the functional properties of IL-21 have not been studied in deep. The main source of IL-21 seems to be represented by activated T cells. IL-21 has been reported to favor both proliferation and maturation of NK cells from bone marrow, in synergy with Flt3-L and IL-15 20. Yet, mice lacking IL-21R display no evident abnormalities in their lymphocyte compartment and no NK cell deficiency 24. Because mice lacking the common γ-chain have a more evident reduction in NK cell numbers 25 than those lacking IL-15Rα 26, it has been speculated that IL-21 could play a role complementary to that of IL-15 in promoting NK cell development in vivo. IL-15 is known to play a central role in driving the NK cell differentiation from CD34+ Lin- precursors in vitro12, 13. Gene targeting experiments clearly indicated that IL-15 is an essential factor for NK cell development in vivo: thus, mice deficient in IL-15 27 or in IL-15R components 25, 26, 28 have profoundly reduced NK cell numbers. Also mice lacking Flt3-L have major NK cell defects, However, they suffer for more evident defects in hematopoiesis, including different hematopoietic lineages (including NK cells) 29.

Other cytokines, such as IL-2 and SCF, would rather play an auxiliary role. Thus, both can synergize with Flt3-L to drive the NK cell development in vitro from bone marrow progenitors12, 16. Although mice lacking IL-2 30 or c-kit 31, 32 have NK cells, these are reduced in number and display an impaired functional activity.

In a previous study we analyzed the progression of cell surface receptor expression in the in vitro-induced human NK cell maturation from CD34+ Lin cell precursors cultured in the presence of cytokines (IL-15, Flt3-L, SCF and IL-7) and stromal cells 19. A remarkable finding was the early expression of major triggering receptors mediating natural cytotoxicity, including NCR and NKG2D. This occurred before the expression of HLA class I-specific inhibitory receptors. Moreover, the appearance of NCR at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. NK cells at early stages of differentiation were also characterized by the surface expression of 2B4 with inhibitory function, i.e.similar to that described previously in mature NK cells from XLP patients 33. This finding suggested that the engagement of 2B4 by its cellular ligand (CD48) could provide a fail-safe mechanism to prevent the unwanted killing of normal autologous cells at the site of NK cell maturation in the BM microenvironment. In these studies, however, NK cells obtained after long-term cultures did not acquire a fully mature surface phenotype since CD2 and CD8 molecules were not expressed, while KIR were expressed either by a very small fraction of developing NK cells or were virtually undetectable.

CD56+ NK cells characterized by little or no expression of CD2 and CD8 were also obtained by Muench et al. 16 upon culture of CD38+ CD34++ Lin precursors in the presence of IL-15, SCF and Flt3-L, and by Mrozek et al. 12 by supplementing CD34+ HPC with IL-15 and SCF. Moreover, both CD34+ CD33 BM progenitors 34 and CD34+ DR BM primitive progenitors 35 cultured in long term cultures supplemented with IL-2 gave rise to a small percentage of cells expressing CD2 and CD8. These data suggested that the complete maturation of human NK cell precursors is a process that requires the cooperation of various cytokines.

Our present data provide novel information on the signals necessary for the differentiation of CD34+ Lin precursors into fully mature NK cells. Indeed, further addition of IL-21 to IL-15, SCF, Flt3-L, and IL-7 allowed developing NK cells to express KIR in proportions similar to those of mature NK cells. In addition, they acquired both CD2 and CD8, two surface moleculescharacteristic of mature NK cells. Remarkably, the NK cell fractions expressing KIR, CD2 and CD8 were only partially overlapping. This suggests that the molecular mechanism involved in the surface expression of these molecules might depend on parallel events generated directly or indirectly by IL-21.

In the presence of IL-21, developing NK cells were also characterized by a bimodal expression of CD56, typical of peripheral blood-derived mature NK cells. In agreement with the phenotypic features of mature NK cells, most KIR+ and CD16+ cells were confined to the CD56dim population, while CD56bright cells expressed higher surface densities of NCR and NKG2A. The CD56bright phenotype is homogeneously expressed by developing NK cells before they reach the "mature" NK phenotype, i.e. when a fraction of these cells becomes CD56dim and acquires KIR and CD16 molecules. This further substantiates the concept 21, 36 that the CD56bright NKG2A+ KIR CD16 (NCRbright) population represents a more immature subset of NK cells as compared to the CD56dim KIR+ CD16+ cells.

Another study reports on the possibility to obtain an efficient NK cell differentiation and KIR expression by developing NK cells 37. In this study, CD34+ Lin CD38 CB cells were cultured with IL-15 or IL-2 in the presence of the murine stromal cell line AFT024. The acquisition of CD94 and KIR required the direct contact between progenitors and AFT024 cells. Based on our present observations it cannot be excluded that AFT024 stromal cells may produce IL-21.

Another interesting finding of our present study was the acquisition of an efficient triggering activity by 2B4. As previously shown, at the early stages of NK cell maturation this molecule functions as an inhibitory receptor and may thus represent a fail safe mechanism to inhibit triggering signals generated by the early-expressed activating receptors (i.e. NCR) 19. Remarkably, in the presence of IL-21, a switch of 2B4 function occurred at later stages of NK cell maturation. The acquisition of the triggering function by 2B4 molecules (as well as by NTB-A that similar to 2B4 requires the association with SH2D1A) by developing NK cells appeared to parallel the expression of a complete repertoire of HLA-specific inhibitory receptors, including NKG2A and KIR. The switch of 2B4 and NTB-A function is likely to depend on the transcription of SH2D1A/SAP molecules, whose appearance could be revealed at late stages of development in NK cell progenitors cultured in the presence of IL-21.

In conclusion, thanks to our present study, it is now possible to recapitulate in vitro the complete process of human NK cell maturation. This may result of great help for a better understanding of the molecular mechanisms leading to the progression of the NK cell development as well as of the mechanisms regulating the expression of the repertoire of the HLA class I-specific inhibitory NK receptors. In addition, the possibility to obtain, within a relatively short time interval, mature NK cells from hematopoietic precursors may be of help in new approaches of adoptive immunotherapy, particularly in diseases, such as acute myeloid leukemias, in which the exploitation of NK cell function has proven highly beneficial to eradicate life-threatening leukemias 38, 39.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Monoclonal antibodies

The following mAb were used in this study: JT3A (IgG2a anti-CD3), c127 (IgG1 anti-CD16), KD1 (IgG2a anti-CD16), c218 (IgG1 anti-CD56), FS280 (IgG2a anti-CD56), A6–220 (IgM anti-CD56), BAB281 (IgG1 anti-NKp46), KL247 (IgM anti-NKp46), AZ20 (IgG1 anti-NKp30), F252 (IgM anti-NKp30), Z231 (IgG1 anti-NKp44), KS38 (IgM anti-NKp44), ON72 and BAT221 (IgG1 anti-NKG2D), MAR206 (IgG1 anti-CD2), XA185 (IgG1 anti-CD94), Z270 (IgG1 anti-NKG2A), 11Pb6 and EB6 (IgG1 anti-KIR2DL1/S1), GL183 (IgG1 anti-KIR2DL2/L3/S2), PAX180 (IgG1 anti-KIR2DS4), FS172 (IgG2a anti-KIR2DS4), Z27 (IgG1 anti-KIR3DL1/S1), Q66 (IgM anti-KIR3DL2), F278 (IgG1 anti-LIR1), PP35 (IgG1 anti-2B4), S39 (IgG2a anti-2B4), MA344 (IgM anti-2B4), MA127 (IgG1 anti-NTB-A),ON56 (IgG2b anti-NTB-A) and B9.4 (IgG2b anti-CD8) were produced in our lab. PE-HPCA2 (anti-CD34) mAb was purchased from Becton Dickinson & Co, Mountain View, CA.

D1.12 (IgG2a anti-HLA-DR) and HP2.6 (IgG2a anti-CD4) mAb were kindly provided by Dr. R. S. Accolla (Università di Insubria, Varese, Italy) and Dr. P. Sanchez-Madrid (Universidad Autonoma de Madrid, Madrid, Spain), respectively.

4.2 Purification of human cord blood CD34+ progenitors and culture conditions

Umbilical cord blood (CB) samples from full term new-born were collected at the Department of Gynecology Istituto Giannina Gaslini (Genova, Italy) upon informed consent of the mothers. CB mononuclear cells (MNC) were obtained by Ficoll-Hipaque density gradient centrifugation. CD34+ cells were separated from MNC using the MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cells obtained in this manner were routinely ≤98% pure. CD34+ Lin cells were cultured in 24-well plates at a concentration of 2×104 in 1.5 ml of MyeloCultH5100 medium (StemCell Technologies Inc., Vancouver, BC) supplemented with 10% human AB serum (ICN Biomedicals, Inc., Irvine, CA), 5% FCS (Euroclone Ltd., GB) and the indicated cytokines. Purified recombinant human IL-15, Flt3-ligand (FL), SCF and IL-7, purchased from Peprotech Inc. (London, GB), and IL-21, purchased from R&D Systems (Minneapolis, MN), were used at 20 ng/ml.

4.3 Generation of polyclonal NK cell populations

Enriched NK cells were isolated by incubating CB mononuclear cells with anti-CD3 (JT3A), anti-CD4 (HP2.6) and anti-HLA-DR (D1.12) mAb (30 min at 4°C), followed by goat anti-mouse-coated Dynabeads (Dynal, Oslo, Norway) (30 min at 4°C) and immunomagnetic depletion. CD3-4-DR cells were cultured on irradiated feeder cells in the presence of 100 U/ml rIL–2 (Proleukin, Chiron Corp., Emeryville, CA) and 1.5 ng/ml PHA (Gibco Ltd, Paisley, Scotland) as described 810.

4.4 Phenotypic analysis and cytolytic activity of NK cells

At regular intervals during culture the phenotype of growing cells was analyzed using a FACScan one- or two-color fluorescence cytofluorimetric analysis 10. Purified CD56+ NK progenitors were tested for cytolytic activity in a 4-h 51Cr -release assay against human (HLA class I FcγR LCL 721.221 cell line), or murine (FcγR+ P815 mastocytoma cell line) targets 8, 9. The concentrations of various mAb added were 0.5 μg/ml for redirected killing or 10 μg/ml for masking experiments. The E/T ratios are indicated in the figure legends.

4.5 RT-PCR analysis

Total RNA was extracted from NK immature precursors or mature NK populations using peQGold RNA pure (peQLab, Erlangen, Germany) and reverse transcribed using oligodT priming. The SH2D1A cDNA (632 bp) was amplified using the following primers: 5′-CAg Cgg CAT CTC CCT Tg (SH2D1A-3 ORF frw) and 5′-TTT CAA AgC TCC TCA CTA Tg (SH2D1A-4 ORF rev). A 228-bp β-actin fragment was amplified as control using the following primers: 5′-ACT CCA TCA TGA AGT GTG ACG (β-actin up) and 5′-CAT ACT CCT GCT TGC TGA TCC (β-actin down). Both amplifications were performed for 30 cycles (30 s at 94°C, 30 s at 60°C, 30 s at 72°C), followed by a 7-min elongation step at 72°C, utilizing AmpliTAQ (Perkin Elmer-Applied Biosystems, Foster City, CA). The PCR products were resolved in a 1.5% agarose gel.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanità (I.S.S.), Ministero della Sanità, Ministero dell'Università e della Ricerca Scientifica e Tecnologica (M.I.U.R.) and Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. Also the financial support of Fondazione Compagnia di San Paolo, Torino, Italy, is gratefully acknowledged. We thank Ms. Tiziana Baffi for secretarial assistance.

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