The proinflammatory cytokines IL-2, IL-15 and IL-21 modulate the repertoire of mature human natural killer cell receptors
© de Rham et al.; licensee BioMed Central Ltd. 2007
Received: 20 July 2007
Accepted: 3 December 2007
Published: 03 December 2007
Natural killer (NK) cells play a crucial role in the immune response to micro-organisms and tumours. Recent evidence suggests that NK cells also regulate the adaptive T-cell response and that it might be possible to exploit this ability to eliminate autoreactive T cells in autoimmune disease and alloreactive T cells in transplantation. Mature NK cells consist of a highly diverse population of cells that expresses different receptors to facilitate recognition of diseased cells and possibly pathogens themselves. Ex vivo culture of NK cells with cytokines such as IL-2 and IL-15 is an approach that permits significant expansion of the NK cell subpopulations, which are likely to have potent antitumour, antiviral, or immunomodulatory effects in autoimmunity. Our data indicate that the addition of IL-21 has a synergistic effect by increasing the numbers of NK cells on a large scale. IL-2 and IL-15 may induce the expression of killer cell immunoglobulin-like receptors (KIRs) in KIR-negative populations, the c-lectin receptor NKG2D and the natural cytotoxic receptor NKp44. The addition of IL-21 to IL-15 or IL-2 can modify the pattern of the KIR receptors and inhibit NKp44 expression by reducing the expression of the adaptor DAP-12. IL-21 also preserved the production of interferon-γ and enhanced the cytotoxic properties of NK cells. Our findings indicate that the proinflammatory cytokines IL-2, IL-15 and IL-21 can modify the peripheral repertoire of NK cells. These properties may be used to endow subpopulations of NK cells with specific phenotypes, which may be used in ex vivo cellular immunotherapy strategies.
Natural killer (NK) cells are an important population of lymphocytes that originally were regarded to play crucial roles in protection from infectious disease and destroying tumour cells; they are also involved in certain autoimmune diseases and in rejection of transplanted tissues [1, 2]. NK cells express many different germline encoded activating or inhibitory receptors that do not rearrange, in contrast to T and B cells, which might suggest that NK cells are unable to respond to more than a limited number of stimuli . The human NK receptors are characterized by genetic diversity, and NK cells were found to express only a subset of these receptors [4, 5]. NK cells can be divided into CD56bright and CD56dim subpopulations, the former being more inclined to produce cytokines such as IFN-γ and the latter to lyse target cells . Several activating and inhibitory NK cell receptors have been well characterized, of which killer cell immunoglobulin-like receptors (KIRs), c-type lectins, and natural cytotoxicity receptors (NCRs). Although inhibitory receptors neutralize NK cells, activating receptors are responsible for NK cell activity [3, 7–9]. The NK repertoire and its modulation at the cell surface is incompletely understood. Many of the activating receptors are constitutively expressed on all NK cells, and it is actually the increased expression of their ligands on other cells induced by mild stimuli that underpins the diversity of activating receptors.
The genetic diversity of NK cell receptors and the range of diseases in which they are thought to play specific roles would suggest that they might potentially be good therapeutic targets. To date, the suitability of KIR as a target of intervention has been suggested by studies of bone marrow transplantation [10–12]. Exploitation of NK alloreactivity may become an important therapeutic strategy in the management of myeloid malignancy, in the modulation of engraftment procedures and in the control of graft-versus-host-disease [13, 14]. In autoimmune disease, NK cells can promote or inhibit the activation of autoimmune T cells, and by virtue of their ability to rapidly kill abnormal cells and produce cytokines and chemokines, NK cells play a key role in regulating autoimmune responses. Human studies and mice models suggest that the immunomodulatory role of NK cells in autoimmunity is likely to provide new insights into the pathogenesis and treatment of autoimmune disorders .
Because NK cells respond to cytokines, and because their killing activity can be enhanced by the presence of IL-2, some investigators have suggested that adaptive transfer of NK cell subsets in an activated state (after stimulation with IL-2, IL-12 and IL-15) may be necessary for optimal efficacy [16, 17]. However, injection of proinflammatory cytokines such as IL-2 or IL-15 to activate endogenous NK cells may induce inflammation and autoreactivity.
Therefore, expansion of NK cells ex vivo is a strategy that is worth considering for several clinical applications. It remains to be determined whether the increase in NK cells ex vivo after exposure to cytokines modifies their peripheral repertoire. In the mouse, cytokines such as IL-2, IL-15, IL-21 and IL-4 can prompt considerable modifications and selective alterations in the repertoire of murine NK cells . In the present study we assessed the effects of IL-2, IL-15 and IL-21 on the human NK cell repertoire, and our findings indicate that addition of IL-21 to IL-2 or IL-15 induced a marked increase in NK cell numbers, and that IL-2 and IL-15 may induce the expression of KIR receptors in a KIR-negative fraction. Our data also indicate that IL-21 can downregulate the expression of NKp44 receptor via the adaptor DAP-12 while preserving production of cytokines by the CD56bright and CD56dim subpopulations of NK cells and even enhancing their cytotoxic function.
Materials and methods
Reagents and cytokines
RPMI-1640 medium, and β-mercaptoethanol were purchased from Sigma Chemicals (St. Louis, MO, USA). Phosphate-buffered saline (PBS), penicillin/streptomycin, L-glutamine, minimal essential medium nonessential amino acids, and sodium pyruvate were supplied by Gibco Invitrogen (San Diego, CA, USA). Human AB serum was provided by the Blood Bank of Geneva University Hospital (Geneva, Switzerland). Ficoll-Paque™ Plus was from Amersham Biosciences (Uppsala, Sweden). Human recombinant (rh)IL-2 was obtained from Biogen Inc. (Cambridge, MA, USA), rhIL-15 was kindly provided by Invitrogen (Seattle, WA, USA) and rhIL-21 was a gift from Dr DC Foster (Zymogenetics, Seattle, WA, USA).
Isolation of NK cells, of CD56dim and CD56brightsubpopulations, and cell cultures
Peripheral blood mononuclear cells (PBMCs) were isolated from normal young donors by density-gradient centrifugation. NK cells were separated from 300 × 106 PBMCs by negative selection using an isolation kit (Miltenyibiotec, Bergisch Gladbach, Germany). Non-NK cells from human PBMCs, such as T cells, B cells, dendritic cells, monocytes, granulocytes and erythrocytes, were stained with a cocktail of biotin-conjugated antibodies to CD3, CD4, CD14, CD15, CD19, CD36, CD123 and CD123a. A second staining was conducted using an antibiotin mAb conjugated with microbeads. The NK cells were isolated by depletion of the magnetically labelled cells. After negative selection, between 3% and 10% NK cells were recovered (depending on the donor). NK cells were washed with PBS and stained with APC-conjugated mAb to CD56 (Miltenyibiotec), biotin-conjugated mAb to CD16 (BD Pharmingen™, San Diego, CA, USA) and APC-Cy7-conjugated mAb to CD3 (BD Pharmingen™). The unlabeled CD16 mAb was stained with streptavidin-ECD, a tandem dye comprising PE covalently linked to Texas-Red (BD Pharmingen™). CD56dim and CD56bright NK cells were subsequently separated on a FACSAria® sorter (BD Pharmingen™). The purity of each subpopulation was consistently greater than 95%. The selected NK cells were cultured at a concentration of 1 × 106 cells/ml for up to 7 days in RPMI medium supplemented with the following: 10% HI AB serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l L-glutamine, 1% minimal essential medium nonessential amino acids and 0.1 mmol/l sodium pyruvate, 5 mmol/l β-mercaptoethanol (at 5 × 10-5 mol/l; referred to as 'medium'). Then, rhIL-2 (25 ng/ml), rhIL-15 (25 ng/ml), rIhL-2 plus rhIL-21 (50 ng/ml), or rhIL-15 plus rhIL-21 was added. The NK cells were placed in 5% carbon dioxide-air humidified atmosphere at 37°C.
Samples of conditioned medium were subjected to enzyme-linked immunosorbent assay for determination of IFN-γ. The sensitivity of all protein assays was 10 to 30 pg/ml. In addition to enzyme-linked immunosorbent assay, an IFN-γ capture assay kit (Miltenyibiotec) was used to determine the amount of IFN-γ. After 7 days of culture with different cytokines, CD56dim and CD56bright NK cells were incubated for 45 min at 37°C together with a bipolar anti-IFN-γ antibody, which binds to the cells as well as to IFN-γ secreted on the cell surface. By using a second antibody, namely PE-conjugated anti-IFN-γ, IFN-γ was determined by fluorescence-activated cell sorting (FACS).
Cell staining for flow cytometry
The following mouse anti-human mAbs were purchased from BD Pharmingen™: PE-Cy7-conjugated anti-CD3; APC-Cy7-conjugated anti-CD16; FITC-conjugated anti-CD158b, which recognizes KIR2DL2 (CD158b1), KIR2DL3 (CD158b2) and KIR2DS2 (CD158j); PE-conjugated anti-CD158a specific for KIR2DL1 (CD158a) and KIR2DS1 (CD158h); biotin-conjugated anti-NKB1 specific for KIR3DL1 (CD158e1); and APC-conjugated anti-NKG2D. PE-conjugated anti-CD56, PE-conjugated anti-CD158i (KIR2DS4) and PE-conjugated anti-NKG2A were supplied by Beckmann Coulter (Fullerton, CA, USA). APC-conjugated anti-CD56, PE-conjugated anti-NKp46, PE-conjugated anti-NKp44 and biotin-conjugated anti-Nkp30 were from Miltenyibiotec, and PE-conjugated anti-NKG2C was from R&D (R&D Systems Inc., Minneapolis, MN, USA). The unlabeled NKB1 mAb was stained with streptavidin-ECD (Beckmann Coulter). For indirect immunofluorescence, nonspecific binding sites were saturated with normal mouse serum before adding the relevant mAb. Six-colour immunofluorescence was performed to assess surface marker expression on CD56dim and CD56bright NK cells activated by rhIL-2, rhIL-15, rhIL-2 plus rhIL-21, or rhIL-15 plus rhIL-21. After 7 days of culture, CD56dim and CD56bright NK cells were washed twice with PBS (completed with 2% foetal calf serum [FCS]) and treated successively with FITC-, PE-, biotin-streptavidin-ECD-, APC-, APC-Cy7-, and PE-Cy7-conjugated mAbs on ice for 10 minutes and washed with PBS (completed with 2% FCS). For the capture assay, NK cells were isolated and washed once with PBS, complemented with 2% FCS. IFN-γ was determined using the capture assay kit from Miltenyibiotec, in accordance with supplier's instructions. Cell staining was analysed using FACSAria® and FACS DIVA™ software (BD Pharmingen™).
CFSE labelling and analysis of NK cell proliferation in vitro
A total of 400 × 106 human PBMCs were labelled with fluorochrome 5-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probe, Inc., Portland, OR, USA), as described previously . CFSE was dissolved in dimethyl sulfoxide and added to the cell suspension for 15 minutes at a final concentration of 0.5 μmol/l at 37°C. The reaction was stopped by the addition of PBS/10% FCS. The cells were washed in PBS/10% FCS, resuspended in RPMI and left to rest overnight at 37°C in a 5% CO2 humid atmosphere. The next day NK cells were isolated from 300 × 106 PBMCs (stained with CFSE) using the NK cell isolation kit (see 'Isolation of NK cells, of CD56dim and CD56bright subpopulations, and cell cultures', above). Then, CD56dim and CD56bright NK cells were separated on a FACSAria® sorter (BD Biosciences PharMingen). The isolated CD56dim and CD56bright NK cells were resuspended in RPMI and cultured at 1 × 106 cells/ml with rhIL-2 (25 ng/ml), rhIL-15 (25 ng/ml), rhIL-2 plus rhIL-21 (50 ng/ml), or rhIL-15 plus rhIL-21 for 5 and 7 days. On days 5 and 7, cells were stained with several conjugated antibodies (see 'Cell staining for flow cytometry', above) and six-colour analysis by flow cytometry was performed on a Becton-Dickinson FACSAria® equipped with FACS DIVA™ software. Live events were collected and analysed by gating on to CD56dim or CD56bright CFSE-positive cells.
Calculation of the frequency of proliferating NK cells
Proliferation of NK cells in response to cytokine stimulation was analyzed as described previously . By means of the FACS acquisition software (FACS DIVA™), the total number of cells in each generation of proliferation was calculated and the number of precursors generating the daughter cells was determined using the formula y/2n, where y is the number of cells in each peak and n is the number of cell divisions. The frequency of NK cell proliferation was then calculated by dividing the total number of precursors by the total number of CFSE-labelled cells.
The isolated NK cells were cultured with rhIL-2 (25 ng/ml), rhIL-15 (25 ng/ml), rhIL-2 plus rhIL-21 (50 ng/ml), or rhIL-15 plus rhIL-21 for 7 days. On day 7, NK cells were subjected to the cytotoxicity assay. To test cytotoxicity, a standard 51Cr-release assay was performed. K562 cells were incubated for 1 hour with Na2 51CrO4 (Hartmann Analytics, Braunschweig, Germany), washed three times and co-incubated for 4 hours with NK effector cells. The percentage of specific lysis was calculated from the following formula: percentage of specific lysis = ([experimental counts – spontaneous lysis]/[maximal lysis – spontaneous lysis] × 100). Experiments were conducted in triplicate.
RNA isolation and real-time PCR
Total cellular RNA was isolated from NK cells by lysing the cells with Qiagen reagent and Qiagen Rneasy® Micro Kit (Qiagen AG, Basel, Switzerland), in accordance with the manufacturer's instructions. One microgram of RNA was treated with DNase to eliminate any contaminating genomic DNA and subsequently reverse transcribed. The quality of the reverse transcription was tested for the expression of the housekeeping gene 18S using real-time polymerase chain reaction (PCR). Subsequently, the relative abundance of KIR genes was determined by TaqMan real-time PCR analysis on an ABI Prism 7300 Sequence detection instrument (Applied Biosystems, Forster City, CA, USA). To quantify the levels of cDNA, the expression of DAP12 and DAP10 was normalized against the housekeeping gene 18S. Data were expressed as relative fold difference between cDNA of the study samples (DAP12 at day 7 with IL-15 and IL-15/IL-21) and a calibrated sample (DAP12 at day 0). DAP12 (Hs00182426_m1), DAP10 (Hs00367159_m1) and 18S (4310893E) primer-probe sets were purchased from Applied Biosystems (Foster City, CA, USA).
Western blot analysis
Purified NK cells were cultured for 7 days with IL-15 (25 ng/ml) or IL-15 plus IL-21 (50 ng/ml). On day 7, cells were harvested and resuspended at 4 × 106 cells/ml in 800 ml of ice-cold PBS and centrifuged. Total cell lysate was prepared and subjected to Western blot analysis. The blots were probed with anti-DAP12 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-tubulin (Sigma). A secondary horseradish peroxidase-conjugated goat anti-rabbit (Dako, Glostrup, Denmark) was added for detection. Antibody-bound proteins were detected by the Uptilight hrp Blot Chemiluminescence substrate (Uptima, Montluçon, France).
Data were analyzed using two-factor analysis of variance test. P < 0.05 and were considered significant (Statview 5.1 [SAS Institute Inc., Cary, NC, USA] and GraphPad Prism 3.02 [GraphPad, Witzenhausen, Germany]).
IL-21 acts in synergy with IL-2 or IL-15 to increase NK cell subpopulations
The KIR repertoire of NK subpopulations after seven days of culture with IL-2 and IL-15 in the presence or absence of IL-21
The KIR phenotype on NK cells stimulated with IL-2, Il-15 and IL-21.
Day 7 + IL-2
Day 7 + IL-15
Day 7 + IL-2/IL-21
Day 7 + IL-15/IL-21
As expected, a higher percentage of CD56bright NK cells failed to express KIR at the cell surface (62.9%) . Of CD56bright NK cells, 17.9% expressed 2DS4 only and around 7.86% of CD56bright expressed at least one of the other KIR (2DL1/S1, 2DL2/L3/S2, or 3DL1) only. 2DS4 and one additional KIR were expressed in 8.33% of the CD56bright cells. 2DS4 plus 2DL1/S1 plus 2DL2/L3/S2 were expressed in 2.38% of the cells, and none of the other combinations of three or four KIR was found at day 0. After 7 days of culture with the different cytokines, the percentage of cells lacking KIR expression increased with IL-21: 62.9% at day 0, 65% and 56% at day 7 with IL-2 and IL-15, respectively, versus 71.55% and 75.24% at day 7 with IL-2/IL-21 and IL-15/IL-21, respectively. Expression by CD56bright cells of two or more KIRs fluctuated more than that of CD56dim (Figure 2b and Table 1), but considering the small percentages of these populations we have refrained from drawing definitive conclusions from these results.
Several donors were tested, and each time between 25% to 45% of CD56dim cells failed to express any KIR, and 35% to 45% expressed predominantly one type of KIR (for example, 2DS4 in Figure 2). In CD56bright between 60% and 85% of the cells did not express any KIR. We did not observe a selective induction of KIR receptors at the cell surface of CD56bright and CD56dim NK cells after 7 days of culture with IL-2 and IL-15 alone or IL-2/IL-21 and IL-15/IL-21, but the fraction of NK cells without any KIR at their surface tended to increase in the presence of IL-21, especially on CD56bright NK cells.
IL-2 and IL-15 induce KIR expression on KIR negative population
It is generally thought that the KIR receptors are acquired by a stochastic mechanism, currently poorly understood, which operates exclusively during NK cell development, and that the repertoire is fixed after maturation. However, in mouse, Gays and coworkers  showed that expression of the Ly49 receptor (the mouse equivalent of KIR in the human system ) can be regulated by cytokines on mature NK cells.
NKp44 expression is induced by IL-2 and IL-15 and down-regulated by IL-21
NK cell subpopulations produce interferon-γ and mediate cytotoxicity after proliferation ex vivoin the presence of cytokines
In the present study, we optimized culture conditions to enhance proliferation of mature human NK cells with IL-2 and IL-15, in the presence or absence of IL-21, and we analyzed the effect that addition of these cytokines had on the NK receptor repertoire of the CD56dim and CD56bright subpopulations of mature NK cells. The main results of this study are as follows. First, IL-21 acts synergistically with IL-2 or IL-15, enhancing markedly the CD56dim NK subpopulation. Second, the KIR repertoire of NK cells was stable in culture, but the KIR-negative cell fraction can express KIR receptors in culture with IL-2 and IL-15, this production being less marked in the presence of IL-21. Finally, IL-21, which is known to downregulate NKG2D, proved also to have the ability to downregulate NKp44 (NCR receptor) induced by IL-15 and to enhance the cytotoxicity of NK cells.
Several experimental studies have demonstrated the capacity of NK cells to eliminate cancer cells. Evidence is now emerging that NK cells might also be a therapeutic target in autoimmunity [2, 26]. Two different strategies could be taken into consideration: the activation of endogenous NK cells or their expansion ex vivo. Taking into account the effects of IL-2, IL-15 and IL-21 on the differentiation, maturation, proliferation and activation of NK cells [9, 27, 28], these cytokines would appear to be particularly suited for manipulating NK cells for therapeutic purpose. Several clinical trials have helped to assess the effect of IL-2 administration on activation and expansion of endogenous NK cells [29, 30]. Recent reports have revealed the effect of IL-21 in preclinical models, suggesting a strong antitumoural activity of IL-21 in renal cell carcinoma, melanoma and leukaemia . However, all three of IL-2, IL-15 and IL-21 have been implicated in autoimmunity, and using such cytokines to activate endogenous NK cells may favour inflammation and promote autoreactivity [9, 27, 28].
Ex vivo adaptive immunotherapy with NK cells has been tested by several groups that have collected and purified clinical grade NK cells before administering patients with doses of up to 107/kg, and IL-2 has already been used to increase numbers of NK cells in a therapeutic approach to melanoma, renal carcinoma cells, or after haematopoietic stem cell transplantation [32–34]. However, it is supposed that small fractions of NK cells characterized by specific phenotypes are responsible for their antiviral, antitumoural, or immunomodulatory activity. In addition, activation by different stimulus or manipulation of NK cell subpopulations by genetic engineering could be much efficient to design NK-cell based immunotherapeutic strategies . Therefore, starting off with a limited number of NK cells would be of great interest for optimizing protocols for increasing the number of NK cells ex vivo by preserving their phenotypes.
Because of their intrinsic effects on NK cells, addition of IL-21 to IL-2 or IL-15 would be the best combination to optimize the ex vivo proliferation of NK cells. Recent data demonstrated that cultured NK cells survived better with IL-15 than with IL-2 in the presence of methylprednisolone, offering interesting clues as to an appropriate NK cell cytokine conditioning regimen in adoptive immunotherapy . In vitro, the effect of IL-21 on the proliferation and differentiation of murine NK cells proved insufficient to drive the proliferation of immature or naïve NK cells; however, at low doses IL-21 enhanced a proliferative response of these cells to either IL-2 or IL-15, whereas high doses had an inhibitory effect . Interestingly, the number of functional NK cells in the peripheral lymphoid organs of IL-21 receptor null mice and the number of bone marrow NK cell precursors are similar in wild-type and γ c-deficient mice, indicating that γ c-dependent cytokines are not required for the earliest commitment events in the NK cell lineage . In addition, Gays and colleagues  demonstrated that IL-21 and combinations of IL-21 and IL-15 or IL-4 can downregulate the expression of the NK gene complex (NKC) and Ly49F receptors after maturation, resulting in an enhanced lytic function. Consequently, in the mouse IL-21 is not essential for NK cell development, but it may influence their proliferation and their maturation into effectors cells.
In human, IL-21 was initially shown to stimulate the development of NK cells in vitro  and is involved in the acquisition of a mature KIR repertoire  from human bone marrow progenitors. IL-21 has also proven crucial to their adoption of a fully functional cytotoxic capacity [31, 37]. On human mature NK cells, IL-21 was recently shown to downregulate NKG2D on NK and T cells stimulated by IL-15 .
Like in the mouse , our results suggest that IL-2 and IL-15 induce similar effects in the human NK cells, both cytokines being able to induce the expression of KIR receptors in a fraction of KIR-negative populations. The addition of IL-21 prevents KIR expression, at least to some extent. The paucity of available KIR mAbs thus represents an obstacle to our study and prevents us from drawing the formal conclusion that the so-called KIR-negative NK cells do not express any activating or inhibitory KIRs. It remains unclear why IL-2 and IL-15, with or without IL-21, failed to modify significantly the KIR repertoire on the KIR-negative subpopulation analysed in NK bulk populations (see Figure 2 and Table 1). The mechanisms underlying the repertoire of a given NK population are not well defined. NK cells do not express all their germline-encoded receptors; instead, a selected combination of these receptors is expressed in a stochastic manner . In the mouse, the pattern of expression of Ly49 receptors is determined by probabilistic transcriptional switches in the promoter regions of the genes and a similar mechanism may exist for KIR . Cytokine environment and infection shape the NK repertoire, which reflects a balance between activating and inhibitory receptors. Signals delivered by cytokines through the JAK/STAT (Janus kiase/signal transducer and activator of transcription) transduction pathway modulate the expression of the different receptors on NK cells to preserve an equilibrium, maintaining self-tolerance and the capacity to produce cytokines or to be cytotoxic. By placing a KIR-negative population in culture, this equilibrium may be significantly modified, resulting in the reactivation or resetting of KIR genes that in turn are susceptible to cytokines.
We confirm recent data reported by Burgess and coworkers ; those investigators reported that IL-21 inhibited DAP10 expression, leading to the downregulation of NKG2D . In addition, we show that IL-21 can inhibit expression of DAP12 (Figure 5). The fact that DAP12 also mediates NKp44 transduction signalling but not that of NKp40 and NKp46  may account for the downregulation of NKp44 by IL-21. Inhibition of DAP12 has several controversial effects. In the mouse DAP12 knockout model, NK cells developed normally but the activating Ly49 receptors were downregulated and nonfunctional . However, recent data also suggest that the absence of DAP12 correlates with potent tumour rejection mediated by NK cell activation . Our results corroborate these data , showing that the NKp44-negative population, which has downregulated DAP12, is more cytotoxic than the NKp44-positive population. Because DAP12 is also required for the expression and signalling of KIR, mainly the KIR activator, we speculate that the reduction of KIR expression observed after 7 days of culture with IL-21 shown in Figure 3b,c (right columns) could be due, at least in part, to the downregulation of DAP12.
In summary, our results strongly suggest that it is possible to modify and ultimately even shape the repertoire of NK cells receptors by modulating ex vivo mature NK cells by means of cytokines. The addition of IL-21 to IL-15 or IL-2 increases significantly the number of cells, with unaltered capacity to produce IFN-γ and even more potent cytotoxic activity, which is quite similar to the effect of IL-21 on murine NK cell biology [18, 41]. With regard to immunotherapy, this insight is fundamental because with this ex vivo strategy it may become possible to envisage tailoring specific phenotypes to subpopulations of NK cell. Human cancer patients would certainly be the first to benefit from a strategy consisting of NK cell based immunotherapy, but one may also envisage, in the near future, treatment of autoimmune diseases after amplifying ex vivo NK cell populations possessing regulatory properties .
= 5-carboxyfluorescein diacetate succinimidyl ester
= DNAX-activating protein of 12 kDa
= fluorescence-activated cell sorting
= foetal calf serum
= killer cell immunoglobulin-like receptor
= monoclonal antibody
= natural cytotoxicity receptor
= natural killer
= NK group 2D
= peripheral blood mononuclear cell
= phosphate-buffered saline
= polymerase chain reaction
= human recombinant.
We are indebted to Dr DC Foster (Zymogenetics, Seattle, WA, USA) for providing human recombinant IL-21 and to Invitrogen for providing human recombinant IL-15.
We are also grateful to Roswitha Rehm for critical reading of the manuscript. This work was supported by Geneva University Hospital, the Department of Internal Medicine, the Dubois-Ferrière-Dinu-Lipatti Foundation (to JV), and the Swiss National Science Foundation, grants Nos 310000-108453 and PMPDA-110347 (to SFL).
- Parham P: MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol. 2005, 5: 201-214. 10.1038/nri1570.View ArticlePubMedGoogle Scholar
- Shi FD, Van Kaer L: Reciprocal regulation between natural killer cells and autoreactive T cells. Nat Rev Immunol. 2006, 6: 751-760. 10.1038/nri1935.View ArticlePubMedGoogle Scholar
- Lanier LL: NK cell recognition. Annu Rev Immunol. 2005, 23: 225-274. 10.1146/annurev.immunol.23.021704.115526.View ArticlePubMedGoogle Scholar
- Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert-Weidenbach K, Corliss B, et al: Human diversity in killer cell inhibitory receptor genes. Immunity. 1997, 7: 753-763. 10.1016/S1074-7613(00)80394-5.View ArticlePubMedGoogle Scholar
- Hsu KC, Chida S, Geraghty DE, Dupont B: The killer cell immunoglobulin-like receptor (KIR) genomic region: gene-order, haplotypes and allelic polymorphism. Immunol Rev. 2002, 190: 40-52. 10.1034/j.1600-065X.2002.19004.x.View ArticlePubMedGoogle Scholar
- Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, Carson WE, Caligiuri MA: Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood. 2001, 97: 3146-3151. 10.1182/blood.V97.10.3146.View ArticlePubMedGoogle Scholar
- Long EO: Regulation of immune responses through inhibitory receptors. Annu Rev Immunol. 1999, 17: 875-904. 10.1146/annurev.immunol.17.1.875.View ArticlePubMedGoogle Scholar
- Biassoni R, Cantoni C, Pende D, Sivori S, Parolini S, Vitale M, Bottino C, Moretta A: Human natural killer cell receptors and co-receptors. Immunol Rev. 2001, 181: 203-214. 10.1034/j.1600-065X.2001.1810117.x.View ArticlePubMedGoogle Scholar
- Colucci F, Caligiuri MA, Di Santo JP: What does it take to make a natural killer?. Nat Rev Immunol. 2003, 3: 413-425. 10.1038/nri1088.View ArticlePubMedGoogle Scholar
- Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, Posati S, Rogaia D, Frassoni F, Aversa F, et al: Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002, 295: 2097-2100. 10.1126/science.1068440.View ArticlePubMedGoogle Scholar
- Ruggeri L, Capanni M, Casucci M, Volpi I, Tosti A, Perruccio K, Urbani E, Negrin RS, Martelli MF, Velardi A: Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood. 1999, 94: 333-339.PubMedGoogle Scholar
- Ruggeri L, Mancusi A, Capanni M, Urbani E, Carotti A, Aloisi T, Stern M, Pende D, Perruccio K, Burchielli E, et al: Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood. 2007, 110: 433-440. 10.1182/blood-2006-07-038687.PubMed CentralView ArticlePubMedGoogle Scholar
- Murphy WJ, Koh CY, Raziuddin A, Bennett M, Longo DL: Immunobiology of natural killer cells and bone marrow transplantation: merging of basic and preclinical studies. Immunol Rev. 2001, 181: 279-289. 10.1034/j.1600-065X.2001.1810124.x.View ArticlePubMedGoogle Scholar
- Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ, et al: Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood. 2005, 105: 3051-3057. 10.1182/blood-2004-07-2974.View ArticlePubMedGoogle Scholar
- Liu R, Van Kaer L, La Cava A, Price M, Campagnolo DI, Collins M, Young DA, Vollmer TL, Shi FD: Autoreactive T cells mediate NK cell degeneration in autoimmune disease. J Immunol. 2006, 176: 5247-5254.View ArticlePubMedGoogle Scholar
- Fehniger TA, Caligiuri MA: Interleukin 15: biology and relevance to human disease. Blood. 2001, 97: 14-32. 10.1182/blood.V97.1.14.View ArticlePubMedGoogle Scholar
- Loza MJ, Perussia B: The IL-12 signature: NK cell terminal CD56+high stage and effector functions. J Immunol. 2004, 172: 88-96.View ArticlePubMedGoogle Scholar
- Gays F, Martin K, Kenefeck R, Aust JG, Brooks CG: Multiple cytokines regulate the NK gene complex-encoded receptor repertoire of mature NK cells and T cells. J Immunol. 2005, 175: 2938-2947.View ArticlePubMedGoogle Scholar
- Lyons AB, Parish CR: Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994, 171: 131-137. 10.1016/0022-1759(94)90236-4.View ArticlePubMedGoogle Scholar
- Kasaian MT, Whitters MJ, Carter LL, Lowe LD, Jussif JM, Deng B, Johnson KA, Witek JS, Senices M, Konz RF, et al: IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity. Immunity. 2002, 16: 559-569. 10.1016/S1074-7613(02)00295-9.View ArticlePubMedGoogle Scholar
- Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, Biassoni R, Moretta L: Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol. 2001, 19: 197-223. 10.1146/annurev.immunol.19.1.197.View ArticlePubMedGoogle Scholar
- Cantoni C, Bottino C, Vitale M, Pessino A, Augugliaro R, Malaspina A, Parolini S, Moretta L, Moretta A, Biassoni R: NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J Exp Med. 1999, 189: 787-796. 10.1084/jem.189.5.787.PubMed CentralView ArticlePubMedGoogle Scholar
- Moretta L, Biassoni R, Bottino C, Mingari MC, Moretta A: Human NK-cell receptors. Immunol Today. 2000, 21: 420-422. 10.1016/S0167-5699(00)01673-X.View ArticlePubMedGoogle Scholar
- Burgess SJ, Marusina AI, Pathmanathan I, Borrego F, Coligan JE: IL-21 down-regulates NKG2D/DAP10 expression on human NK and CD8+ T cells. J Immunol. 2006, 176: 1490-1497.View ArticlePubMedGoogle Scholar
- Takaki R, Watson SR, Lanier LL: DAP12: an adapter protein with dual functionality. Immunol Rev. 2006, 214: 118-129. 10.1111/j.1600-065X.2006.00466.x.View ArticlePubMedGoogle Scholar
- Li Z, Lim WK, Mahesh SP, Liu B, Nussenblatt RB: Cutting edge: in vivo blockade of human IL-2 receptor induces expansion of CD56(bright) regulatory NK cells in patients with active uveitis. J Immunol. 2005, 174: 5187-5191.View ArticlePubMedGoogle Scholar
- Becknell B, Caligiuri MA: Interleukin-2, interleukin-15, and their roles in human natural killer cells. Adv Immunol. 2005, 86: 209-239. 10.1016/S0065-2776(04)86006-1.View ArticlePubMedGoogle Scholar
- Leonard WJ, Spolski R: Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation. Nat Rev Immunol. 2005, 5: 688-698. 10.1038/nri1688.View ArticlePubMedGoogle Scholar
- Rosenberg SA: Interleukin-2 and the development of immunotherapy for the treatment of patients with cancer. Cancer J Sci Am. 2000, S2-S7. Suppl 1
- Farag SS, Caligiuri MA: Cytokine modulation of the innate immune system in the treatment of leukemia and lymphoma. Adv Pharmacol. 2004, 51: 295-318.View ArticlePubMedGoogle Scholar
- Curti BD: Immunomodulatory and antitumor effects of interleukin-21 in patients with renal cell carcinoma. Expert Rev Anticancer Ther. 2006, 6: 905-909. 10.1586/1473718.104.22.1685.View ArticlePubMedGoogle Scholar
- Igarashi T, Wynberg J, Srinivasan R, Becknell B, McCoy JP, Takahashi Y, Suffredini DA, Linehan WM, Caligiuri MA, Childs RW: Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells. Blood. 2004, 104: 170-177. 10.1182/blood-2003-12-4438.View ArticlePubMedGoogle Scholar
- Koehl U, Esser R, Zimmermann S, Tonn T, Kotchetkov R, Bartling T, Sörensen J, Grüttner HP, Bader P, Seifried E, et al: Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children. Klin Padiatr. 2005, 217: 345-350. 10.1055/s-2005-872520.View ArticlePubMedGoogle Scholar
- Passweg JR, Stern M, Koehl U, Uharek L, Tichelli A: Use of natural killer cells in hematopoetic stem cell transplantation. Bone Marrow Transplant. 2005, 35: 637-643. 10.1038/sj.bmt.1704810.View ArticlePubMedGoogle Scholar
- Ljunggren HG, Malmberg KJ: Prospects for the use of NK cells in immunotherapy of human cancer. Nat Rev Immunol. 2007, 7: 329-339. 10.1038/nri2073.View ArticlePubMedGoogle Scholar
- Chiossone L, Vitale C, Cottalasso F, Moretti S, Azzarone B, Moretta L, Mingari MC: Molecular analysis of the methylprednisolone-mediated inhibition of NK-cell function: evidence for different susceptibility of IL-2-versus IL-15-activated NK cells. Blood. 2007, 109: 3767-3775. 10.1182/blood-2006-07-037846.View ArticlePubMedGoogle Scholar
- Parrish-Novak J, Dillon SR, Nelson A, Hammond A, Sprecher C, Gross JA, Johnston J, Madden K, Xu W, West J, et al: Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature. 2000, 408: 57-63. 10.1038/35040504.View ArticlePubMedGoogle Scholar
- Sivori S, Cantoni C, Parolini S, Marcenaro E, Conte R, Moretta L, Moretta A: IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors. Eur J Immunol. 2003, 33: 3439-3447. 10.1002/eji.200324533.View ArticlePubMedGoogle Scholar
- Alves NL, Arosa FA, van Lier RA: IL-21 sustains CD28 expression on IL-15-activated human naive CD8+ T cells. J Immunol. 2005, 175: 755-762.View ArticlePubMedGoogle Scholar
- Valiante NM, Uhrberg M, Shilling HG, Lienert-Weidenbach K, Arnett KL, D'Andrea A, Phillips JH, Lanier LL, Parham P: Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity. 1997, 7: 739-751. 10.1016/S1074-7613(00)80393-3.View ArticlePubMedGoogle Scholar
- Saleh A, Davies GE, Pascal V, Wright PW, Hodge DL, Cho EH, Lockett SJ, Abshari M, Anderson SK: Identification of probabilistic transcriptional switches in the Ly49 gene cluster: a eukaryotic mechanism for selective gene activation. Immunity. 2004, 21: 55-66. 10.1016/j.immuni.2004.06.005.View ArticlePubMedGoogle Scholar
- Bakker AB, Hoek RM, Cerwenka A, Blom B, Lucian L, McNeil T, Murray R, Phillips LH, Sedgwick JD, Lanier LL: DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity. 2000, 13: 345-353. 10.1016/S1074-7613(00)00034-0.View ArticlePubMedGoogle Scholar
- Bielekova B, Catalfamo M, Reichert-Scrivner S, Packer A, Cerna M, Waldmann TA, McFarland H, Henkart PA, Martin R: Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis. Proc Natl Acad Sci USA. 2006, 103: 5941-5946. 10.1073/pnas.0601335103.PubMed CentralView ArticlePubMedGoogle Scholar
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