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International Immunology Advance Access originally published online on November 15, 2005
International Immunology 2006 18(1):49-58; doi:10.1093/intimm/dxh348
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© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Effect of IL-21 on NK cells derived from different umbilical cord blood populations

Sonia A. Perez1, Louisa G. Mahaira1, Panagiota A. Sotiropoulou1, Angelos D. Gritzapis1, Eleni G. Iliopoulou1, Dimitrios K. Niarchos1, Nike T. Cacoullos1, Yannis G. Kavalakis2, Aris I. Antsaklis2, Nectaria N. Sotiriadou1, Constantin N. Baxevanis1 and Michael Papamichail1

1 Cancer Immunology and Immunotherapy Center, Saint Savas Hospital, 171 Alexandras Avenue, Athens 115 22, Greece
2 1st Obstetrics and Gynecology University Clinic, Alexandras Maternity Hospital, Athens, Greece

Correspondence to: S. Perez; E-mail: perez{at}ciic.gr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
IL-21 plays a role in the proliferation and maturation of NK cells developed from hematopoietic stem cells. In this study, we found that IL-21, in the presence of physiological concentration of hydrocortisone (HC), has a significant impact on the functions of NK cells derived from umbilical cord blood (CB) populations. We demonstrate that IL-21, in combination with Flt3-ligand, IL-15 and HC, induces high proliferative responses and, apart from enhancing NK-mediated cytotoxicity, it also induces a significant increase in lymphokine-activated killer activity of CB/CD34+-derived CD56+ cells. In addition, IL-21 induced changes in the CD56+ cell cytokine secretion profile. Thus, we observed increased levels of IL-10 and granulocyte macrophage colony-stimulating factor, whereas tumor necrosis factor-{alpha} levels decreased. IFN-{gamma} production was also modified by IL-21, depending on the presence or absence of IL-18. CB/CD34+ cells did not express the IL-21R ex vivo, but receptor expression was induced during their commitment to differentiation into CD56+ cells. Our data ascribe to IL-21 an essential role on NK cell development and function under conditions similar to the in vivo CB microenvironment.

Keywords: CD34, CD56, hydrocortisone, IL-21R


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
IL-21 is a recently described cytokine with homology to IL-2 and IL-15 (1). The IL-21-specific receptor is closely related to the IL-2R ß-chain and the IL-4R {alpha}-chain (2). IL-21R shares the {gamma}-chain with IL-2R, IL-4R, IL-7R, IL-9R and IL-15R (3). Recent studies in the literature demonstrate that IL-21 influences lymphoid cell development and function. Indeed, in vitro studies have shown that IL-21 activates human peripheral blood NK cells (1), induces the terminal differentiation of mature mouse NK cells (4) and potentiates the development of mature NK cells from both murine (5) and human (1) bone marrow (BM) cells, as well as from human cord blood (CB) CD34+ precursors (6). In addition, IL-21 in synergism with granulocyte macrophage colony-stimulating factor (GM-CSF) is able to modulate differentiation and maturation of myeloid BM dendritic cells (7). IL-21 is also involved in the promotion of antigen-specific CD8+ T cell-mediated responses (8), in the modulation of B cell responses (9, 10) and has a differential effect on the regulation of Th1 responses (11, 12). Thus, IL-21 plays an important role in immune regulation by modulating the response of effector lymphocytes depending on the antigenic stimulus. Moreover, recent reports (13–15) have ascribed anti-tumor effects to IL-21 since in vivo administration of IL-21, either by transduction of the IL-21 gene into human and murine cell lines or by direct gene delivery (16–18), produced potent tumor cell-directed innate and adaptive immunities.

We have recently reported that NK cells can develop from CB/CD34+ in the presence of physiological levels of hydrocortisone (HC) (19) and that HC enhances NK cell proliferation and cytokine secretion (20). Given the fact that IL-21, along with Flt3-ligand (FL) plus IL-15, has been reported to support the in vitro proliferation and differentiation of human CD34+ hematopoietic stem cell (HSC) into mature NK cells (1, 6), we sought to investigate the effect of IL-21 on NK development from CB/CD34+ progenitors in the presence of HC, thereby mimicking the in vivo milieu of CB, which contains active cortisol (21). We also analyzed the effect of IL-21, under the same conditions, on CB/CD56+ cells. Cytokine secretion profiles and cytotoxic effector functions of the above cell populations, as well as quantitation of IL-21R mRNA levels on cytokine-induced CB cells, were also investigated. We show that IL-21, in the presence of HC, further increases the proliferative capacity and the cytotoxic potential of NK cells derived from CB/CD34+. In addition, our results demonstrate that IL-21, in combination with HC, modulates the IL-10, GM-CSF, tumor necrosis factor-{alpha} (TNF-{alpha}) and IFN-{gamma} secretion profile of CB/CD56+ and CB/CD34-derived NK cells, thereby emphasizing the prominent immunoregulatory role of IL-21, under conditions reflecting the in vivo microenvironment. Lastly, we demonstrate, for the first time, that freshly isolated CB/CD34+ do not express IL-21R, either at the mRNA or the protein level, but they do so when cultured with FL or FL/IL-21, even in the absence of IL-15. Furthermore, we show that IL-21R is only transiently expressed at high levels on the cell surface, before CB/CD34+ cells acquire CD56 antigen expression. CB/CD34-derived CD56+ cells express very low levels of surface IL-21R as their freshly isolated or cultured CB/CD56+ counterparts.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
CB samples and mononuclear cell purification
Samples of human CB were obtained from umbilical veins of normal full-term infants after informed consent of the mothers. The cells were processed within 4 h of collection. CB samples were diluted 1:2 in HBSS (Life Technologies Ltd, Paisley, Scotland). Mononuclear cells were isolated by Ficoll-Hypaque centrifugation using standard procedures. CD34+ cells were isolated with a CD34-selection kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD56+, CD3+, CD14+ and CD19+ cells were isolated from the CD34– fraction using anti-CD56, CD3, CD14 or CD19 microbeads (Miltenyi Biotec), respectively. CD34+ cells were further purified by cell sorting of CD34+ cells negative for CD56, CD3, CD7, CD14 and CD19 expression, with a Coulter Epics Altra cell sorter (Beckman Coulter, Fullerton, CA, USA).

Cell lines
The human cell lines K562, Daudi, Raji and U937 were obtained from the American Type Culture Collection (Manassas, VA, USA). The human melanoma cell line FM3 (22) was provided by J. Zeuthen (Department of Tumor Cell Biology, Danish Cancer Society Research Center, Copenhagen). NK-92 cells were obtained from DSMZ GmbH (Braunschweig, Germany). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 50 µg ml–1 gentamicin (all purchased from Life Technologies) at 37°C in a CO2 incubator. For NK-92 growth, IL-2 (Proleukin, Chiron Corp., Emeryville, CA, USA) at 10 ng ml–1 was added to the culture medium.

Cell cultures
Depending on the cell number of the starting population (5 x 104 cells or 5 x 105 cells per culture), cultures were grown in 0.1 or 1.0 ml medium in 96- or 24-well culture plates, respectively. MyeloCult (Stem Cell Technologies, Vancouver, British Columbia, Canada) supplemented with 10–6 M freshly dissolved HC (Sigma, St Louis, MO, USA) and 50 µg ml–1 gentamicin was used as culture medium. FL and IL-15 (R&D Systems, Abingdon, UK) were added at 25 and 20 ng ml–1, respectively. IL-21 (ZymoGenetics, Seattle, WA, USA) was used at 30 ng ml–1. Every 3–4 days, half of the medium was discarded and replenished by fresh medium containing freshly added cytokines. Cell density was thereby adjusted to 0.5 x 106 cells ml–1.

Antibodies and immunophenotyping
mAbs, anti-CD34 conjugated with PE and anti-CD56 conjugated with FITC, were obtained from Becton Dickinson (Mountain View, CA, USA). Anti-CD14, CD3, CD7, CD19 and anti-2B4 conjugated with FITC and anti-NKG2D, NKp46 and perforin mAbs conjugated with PE were purchased from PharMingen (San Diego, CA, USA). PE–cytochrome 5-conjugated anti-CD56 and anti-CD16, CD161, CD94, CD158a, CD158b labeled with PE were obtained from Immunotech (Marseille, France). Anti-IL-21R–PE-conjugated mAb was obtained from R&D Systems. For cell-surface staining, cells were washed twice with ice-cold PBS/1% BSA/NaN3 followed by incubation with saturating concentrations of the appropriate mAbs for 15 min at room temperature. Thereafter, cells were washed twice in ice-cold PBS/1% BSA/NaN3 and fixed with 1% PFA in PBS. For intracellular staining, cells were fixed and rendered permeable using the Fix and Perm kit from Caltag Medsystems (Buckingham, UK), according to the manufacturer's instructions. Samples were analyzed using FACSCalibur (Becton Dickinson, Heidelberg, Germany) and CellQuest analysis software.

Cytotoxicity assay
Cytotoxic activity of cultured cells was determined in a standard 4-h 51Cr-release assay against the NK-sensitive cell line K562 and the NK-resistant cell lines Daudi, Raji, U937 and FM3 as previously described (19). In brief, target cells were labeled with 100 µCi sodium [51Cr] chromate (Radiochemical Centre, Amersham, Bucks, UK) per 106 target cells for 1 h. Effector cells were incubated with target cells at the indicated ratios. Spontaneous 51Cr release was measured by incubating target cells in the absence of effector cells. Maximum 51Cr release was determined by adding 1% Triton X-100 (Sigma). Spontaneous lysis did not exceed 10% of the maximum release. The amount of 51Cr released was measured in a {gamma}-counter (Packard, Downers Grove, IL, USA) and the percent lysis was calculated as follows: % specific lysis = [(experimental 51Cr release – spontaneous 51Cr release)/(maximum 51Cr release – spontaneous 51Cr release)] x 100.

Quantitation of cytokines in culture supernatants
For cytokine production determinations, a modification of the protocol reported by Cooper et al. (23) was applied. In brief, cells recovered from cultures with FL and IL-15, with or without IL-21, were washed twice with HBSS and incubated for 48 h in fresh medium containing FL (25 ng ml–1), IL-15 (20 ng ml–1) and IL-12 (2 ng ml–1) (R&D Systems), with or without IL-18 (100 ng ml–1) (R&D Systems), in the presence or absence of IL-21 (30 ng ml–1). For IL-10 neutralization, a mAb against human IL-10 (clone 25209 from R&D Systems) was also included in the culture medium at a final concentration of 10 µg ml–1. Supernatants were collected by centrifugation and stored at –70°C until use. Cytokines were quantitated using commercially available ELISA kits (for IFN-{gamma}, TNF-{alpha} and IL-10 from Diaclone Research, Besancon, France and for GM-CSF from R&D Systems) according to the manufacturer's instructions.

IL-21R mRNA detection and quantitation by reverse transcription–PCR and real-time PCR
Total RNA was extracted from cells either freshly isolated from CB sub-populations or cultured as described previously in Cell Cultures and from established human cell lines, using the SV Total RNA Isolation System (Promega, Charbonnieres, France), according to the manufacturer's protocol. First strand cDNA synthesis was performed using ~1 µg of total RNA, random primers and the SuperScript III RNase H (–) reverse transcriptase (Invitrogen, Paisley, Scotland). Two microliters of this cDNA was used for PCR amplification with Taq Platinum (Invitrogen) and primers specific for human IL-21R, that were designed using Primer3 Software (Whitehead Institute and Howard Hughes Medical Institute, http://www.broad.mit.edu) under the following conditions: denaturation at 94°C for 5 min, followed by amplification rounds consisting of 94°C for 1 min, 60°C for 1 min and 72°C for 1.5 min, for 45 cycles.

The sequences of the primers used are: IL-21R sense: 5'-GCGCTCAGATTACGAAGACC-3' and IL-21R antisense: 5'-GAGTCTTTGCGGAACTCCAG-3'.

The reverse transcription (RT)–PCR control of the integrity of the cDNA was ß2-microglobulin amplified under the same PCR parameters using the following set of primers: ß2-microglobulin sense: 5'-GTCTGGGTTTCATCCATCCG-3' and ß2-microglobulin antisense: 5'-TCATCCAATCCAAATGCGGC-3'.

cDNA from K562 cells was used as a negative control and NK-92 cells as positive control. All the amplified products were subjected to 2% agarose gel electrophoresis containing GelStar dye (FMC BioProducts, Rockland, ME, USA) and visualized by UV light.

To study the mRNA levels of the receptor and how the presence of the cytokine affected them, we performed real-time PCR experiments. Two microliters of the synthesized cDNA were used as a template for the reaction, which was carried out using Platinum Quantitative PCR SybrGreen SuperMix-UDG (Invitrogen), in a Rotor-Gene 3000 machine (Corbett Research, Sydney, Australia), according to the manufacturer's recommendations. For real-time PCR experiments, a second set of primers was designed, also using Primer3 Software, with the following sequence: IL-21R sense: 5'-TATCTCCTGGCGCTCAGATT-3' and IL-21R antisense: 5'-GAGTCTTTGCGGAACTCCAG-3'. The cycling protocol consisted of 50°C for 2 min and 95°C for 2 min (uracil-DNA glycosylase reaction) and 50 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 10 s, heating at 78°C (to exclude any irrelevant fluorescence from primer dimmers) and plate reading. To confirm amplification specificity, we performed melting curve analysis at the end of each cycling program. ß2-microglobulin was used as a housekeeping gene and was amplified using the same set of primers mentioned above, in the same thermal protocol with IL-21R. To evaluate the relative amount of transcripts in each sample, the Ct value of the housekeeping gene was subtracted from the Ct of the target gene (={Delta}Ct). {Delta}Ct values were normalized by subtracting the {Delta}Ct value of an internal control sample (freshly isolated adult CD56+ cells, which was used in each run) from the {Delta}Ct value of each sample (={Delta}{Delta}Ct) and the exported number was used in the equation 2–{Delta}{Delta}Ct (=IL-21R arbitrary units).

Limiting dilution analysis
For the limiting dilution analysis (LDA) assay, purified CB/CD34+ cells (>98% purity) were cultured for 10 days with FL or FL/IL-21, washed and then seeded in 96-well, U-bottomed plates (16 replicates per cell concentration) at dilutions ranging from 5000 to 25 cells per well in the presence of IL-15 alone. HC 10–6 M was always included in the culture medium. Half of the medium was renewed every 3–4 days. After 20 days, the wells containing viable cells were tested for CD56 expression by flow cytometry. NK progenitor frequency was calculated as the reciprocal of the concentration of cells that resulted in 37% negative wells using Poisson statistics and the weighted mean method (24, 25).

Statistical analysis
Statistically significant differences in the parameters tested in CB-derived CD56+ cells cultured in the presence or absence of IL-21 were assessed by applying Student's t-test statistics.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Effect of IL-21 on the expansion of NK cells from different CB populations
We first tested the effect of IL-21, in combination with FL, IL-15 and HC on the development of NK cells from purified CB/CD34+ cells, which have been demonstrated to give rise to NK cells when cultured with FL and IL-15 in the presence of 10–6 M HC (17), as well as on CB-isolated CD56+ cells. Cells were collected at regular time points (i.e. 5- to 10-day intervals) after culture initiation to assess cell proliferation and CD56 expression. When FL/IL-15/HC-containing media were supplemented with IL-21, a significant increase (P < 0.01) was observed in CD56+ cell numbers (Fig. 1), which paralleled the increase in the total cell proliferation rates (data not shown). This effect was already detectable 15 days after culture initiation and was seen with both starting populations (i.e. CD56+ and CD34+ cells). The outgrowth, however, of NK cells derived from CB/CD34+ cells, (320 x 106 versus 910 x 106 CD56+ cells without or with IL-21, respectively), was significantly higher (P < 0.01) than those derived from CB/CD56+ cells, (115 x 106 versus 248 x 106 CD56+ cells), irrespective of the cytokine cocktail used. In accordance with earlier reports (1), none of the two CB sub-populations proliferated in the presence of IL-21 alone (data not shown), suggesting that this cytokine had no direct effect on the expansion of CB cells. IL-15 alone or in combination with IL-21, in the absence of FL, could not support CB/CD34+ cell proliferation. This is in agreement with previous studies demonstrating that CB/CD34+ cells are not responsive to IL-15 since they do not express IL-15R (26). On the contrary, CB/CD56+ cells responded similarly to IL-15 and IL-15/IL-21 as they did to FL/IL-15 and FL/IL-15/IL-21 (data not shown).



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  		Fig. 1. IL-21 induces proliferation in CB-derived CD56+ populations. CB/CD34+ (A), and CB/CD56+ (B) cells were cultured for 30 days in media supplemented with FL/HC in the presence or absence of IL-15, with or without IL-21, as indicated. Results are presented as mean values ± SD from five individually performed experiments.

 
Furthermore, although FL alone, it did not support the growth of CB/CD56+ cells, it induced the proliferation of CB/CD34+ cells, as expected (26) and the addition of IL-21 only slightly enhanced their proliferative rate (Fig. 1A).

Effect of IL-21 on NK and lymphokine-activated killer activity of CD56+ cells derived from different CB populations
We subsequently examined the effector function of CD56+ cells derived from CB/CD34+ and CB/CD56+ cell populations in the presence of IL-21 and FL, IL-15 and HC and compared it with the lytic activity of CD56+ effectors derived from the same CB populations in the presence of FL, IL-15 and HC alone. The presence of IL-21 significantly increased NK cytotoxicity in CB/CD34-derived CD56+ cells; this effect was more intense in less differentiated NK cells (harvested on day 15, P < 0.01) than in more mature NK cells tested by culture termination (day 30, P < 0.05) (Fig. 2B). No statistically significant changes could be detected with NK effectors from day 15 and day 30 CB/CD56+ cells (Fig. 2A). The most prominent effect induced by IL-21 was seen when testing CD56+ effectors derived from CB/CD34+ cells against the lymphokine-activated killer (LAK)-sensitive Daudi targets. There was a remarkable increase in LAK cytotoxicity with 30-day cultured CB/CD34-derived CD56+ effectors, but not with those grown for only the first 2 weeks (Fig. 2D). This demonstrated that only NK effectors that had matured under the driving force of IL-21, in combination with FL and IL-15, in the presence of HC, could exert this lytic activity. The increase in cytotoxicity against Daudi cells was not unique for this type of tumor targets, but for several other LAK-sensitive tumor targets such as Raji (Burkitt lymphoma), U937 (histiocytic lymphoma) and FM3 (melanoma) (Fig. 3). As for NK cytotoxicity, IL-21 did not statistically increase LAK activity in CB/CD56+ effectors (Fig. 2C).



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  		Fig. 2. Effect of IL-21 on NK and LAK activities of CB-derived NK cells. CB/CD56+, and CB/CD34+ cells were cultured for 15 or 30 days in the presence of FL/IL-15/HC, with or without IL-21, and tested for cytotoxic activity, at the indicated effector:target (E:T) ratios, against K562 (A, B) and Daudi (C, D) targets. Results are represented as the mean values ± SD of four independently performed experiments. Each experiment was conducted with different CB sample.

 


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  		Fig. 3. Effect of IL-21 on LAK activity of CB/CD34+-derived NK cells against various target cell lines. CB/CD34+ cells were cultured for 30 days in the presence of FL/IL-15/HC, with or without IL-21 and tested for cytotoxic activity against Daudi, Raji, FM3 and U937 targets, at effector:target (E:T) ratio of 5. Three experiments, involving different CB samples, were performed and the mean values ± SD from these results are shown.

 
The acquisition of cytotoxic function by maturing NK cells is related to the appearance of a series of receptors on effector cells. In order to investigate if the increased cytotoxic activity of NK cells derived from the CB/CD34+ population in the presence of IL-21 could be attributed to differences in NKRs, we examined the expression of CD16, CD161, CD94, CD158a, CD158b, NKG2D, NKp46 and 2B4 in CB-derived NK cells cultured for 30 days in the presence or absence of IL-21. As shown in Table 1, no statistically significant differences could be observed in any of the examined receptors. As the killing capacity of NK cells is also related to perforin expression, we also examined the levels of intracellular perforin in CB-derived NK cells, in the presence or absence of IL-21. In this case as well, no statistically significant differences could be detected (Table 1). These results suggest that other molecules than the ones assayed above, may be implicated to the increased cytotoxic potential of NK cells derived from CB/CD34+ cells in the presence of IL-21 (i.e. adhesion molecules and granzymes).


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  		Table 1. Phenotypic characterization of NK cells derived from different CB subsets after 30 days of culture in FL/IL-15 in the presence or absence of IL-21

 
Effect of IL-21 on cytokine production potential of CD56+ cells derived from different CB populations
We have recently reported that mature NK cells derived from CB/CD34+ cells, in the presence of FL and IL-15, can produce IFN-{gamma}, TNF-{alpha}, GM-CSF and IL-10 (19). To test whether IL-21 influences the cytokine production potential in this system, NK cells harvested from 30-day cultures initiated in the presence of FL, IL-15 and HC, with or without IL-21, were further incubated for 48 h with IL-12 and IL-18. The latter are known to induce secretion of a series of cytokines by NK cells (23, 27), including the ones mentioned above. IL-21 positively influenced GM-CSF production by NK cells derived from both CB populations (Fig. 4A). This effect was much more evident with NK cells originating from the CB/CD34+ population (P < 0.01) which in the absence of IL-21 produced only minimal levels of GM-CSF. There was also a statistically significant increase in the levels of GM-CSF produced by CB/CD56+ cells (P < 0.05) which, even in the absence of IL-21, secreted fairly high levels of this cytokine. In contrast to GM-CSF, we found that TNF-{alpha} production by NK effectors derived from the two types of CB populations was significantly reduced (P < 0.01) in the presence of IL-21 (Fig. 4B). IL-10 has been previously reported to inhibit TNF-{alpha} production (28, 29). In order to examine whether the observed decrease of TNF-{alpha} production in the presence of IL-21 could be attributed to the increased levels of IL-10, we used a neutralizing antibody against IL-10 to inhibit its bioactivity. IL-10 neutralization did not reverse the inhibitory effect of IL-21 on TNF-{alpha} production.



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  		Fig. 4. Effect of IL-21 on cytokine production by CB-derived NK cells. CB/CD56+ (CD56) and CB/CD34+ (CD34) cells were cultured for 30 days in the presence of FL/IL-15/HC, with or without IL-21. For the last 48 h of culture, IL-12 and IL-18 and, where indicated, IL-21 were added to the media. For inhibition of the endogenously produced IL-10, a neutralizing anti-IL-10 mAb was also included in the culture. Supernatants were assayed for GM-CSF, TNF-{alpha} and IL-10 production. Values are expressed as pg ml–1 of the respective cytokine per 105 cells. The data represent the mean values ± SD from three experiments with different CB samples.

 
We have recently shown (19) that NK cells collected from 15- and 30-day cultures of CB/CD34+ cell populations, set up with FL and IL-15 in the presence of HC, produce low levels of IL-10. In the present report, we also found fairly low levels of IL-10 produced by these cells, which were significantly increased by the co-addition of IL-21 (Fig. 4C). In contrast to CB/CD34-derived NK cells, those originating from CB/CD56+ cells produced much higher levels of IL-10 in cultures with FL, IL-15 and HC, which further increased in the presence of IL-21 (Fig. 4C).

The presence of IL-12 plus IL-18 for 48 h in cultures stimulated IFN-{gamma} secretion by NK cells derived from different CB sources. As shown in Fig. 5(A), there was no statistically significant change in IFN-{gamma} production in the presence of IL-21 by NK cells derived from both CB cell populations. Given the previous report by Goodier and Londei (30), demonstrating a negative effect of IL-10 on IFN-{gamma} production by NK cells, it was somewhat peculiar not to detect decreased IFN-{gamma} production caused by the IL-21-induced endogenous IL-10. A possible explanation for this might be that the strong synergistic effect of IL-12 and IL-18 on IFN-{gamma} production (31) may interfere with any inhibitory effect mediated by IL-10. To address this question, we set up similar cultures by adding only IL-12 for the last 48 h of incubation. Indeed, in the absence of exogenous IL-18, IFN-{gamma} levels were reduced in cultures with increased production of IL-10 (Fig. 5B). In these cultures, neutralization of IL-10 reversed the IL-21-mediated reducing effect on IFN-{gamma} production.



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  		Fig. 5. Effect of IL-21 on IFN-{gamma} production by CB-derived NK cells. CB/CD56+ and CB/CD34+ cells were cultured for 30 days in the presence of FL/IL-15/HC, with or without IL-21. For the last 48 h of culture, media were supplemented with IL-12 and IL-18 (A) or IL-12 (B), in the presence or absence of IL-21 and/or anti-IL-10 mAb, as indicated. Supernatants were assayed for IFN-{gamma} production. Values are expressed as pg ml–1 of IFN-{gamma} per 105 cells. The data represent the mean values ± SD from three experiments conducted with separate CB samples.

 
Both CB-derived NK cell populations produced very low amounts of IL-4 or IL-13 (<10 pg ml–1) when cultured either in the presence of FL/IL-15/HC or FL/IL-15/HC/IL-21 (data not shown).

To address the question whether IL-21 directly affects cytokine production as has been previously reported for IFN-{gamma} (12, 32), we measured the cytokine production of cells grown for 30 days in the presence of FL/IL-15, after a 48-h incubation with IL-21 in combination with IL-12/IL-18. This relatively short exposure to IL-21 could not induce significant differences in the cytokine profile of both CB-derived NK cell populations (Fig 4 and 5A).

IL-21R expression in CB-derived NK cells
Our data so far raise the question as to whether IL-21-induced effects on CD56+ cells derived from different CB sub-populations, require the expression of the specific IL-21R. To address this question, we tested for IL-21R mRNA expression in the CB sub-populations both ex vivo and after culture with FL/IL-15 in the presence or absence of IL-21. As shown in Fig. 6(A), IL-21R mRNA was not detectable in ex vivo isolated CB/CD34+, whereas it was present in CB/CD56+ cells.



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  		Fig. 6. (A) IL-21R mRNA detection by RT–PCR in freshly isolated cell populations from CB samples. Panel I presents the IL-21R product and panel II the ß2-microglobulin product of the same samples, used to monitor mRNA integrity. MW: DNA molecular weight markers. The NK-92 cell line was used as a positive control and K562 cells as a negative control. (B) IL-21R mRNA expression in CB/CD56+ and CB/CD34+ cells cultured for 28 days with FL and/or FL/IL-15, in the presence or absence of IL-21. Values are presented as mean ± SD of the measured arbitrary units estimated by the Ct values of each sample, normalized with the respective Ct value of the reference gene and using a standard curve.

 
To follow IL-21R mRNA expression in CD34+ and CD56+ cells during their proliferation and/or differentiation, we set up cultures of CB/CD34+ with FL/HC or FL/IL-15/HC, in the presence or absence of IL-21 and CB/CD56+ cells, only with FL/IL-15/HC or FL/IL-15/HC/IL-21; in the absence of IL-15 the growth of these cells is not supported.

As shown in Fig. 6(B), IL-21R mRNA expression in CB/CD56+ cells, either in the presence or absence of IL-21, was not significantly altered during the 28-day culture period.

On the other hand, IL-21R mRNA expression was induced in cultured CB/CD34+ cells. A progressive increase was observed in all cultures, up to day 20. After that time point, cultures grown in the absence of IL-15 (lacking CD56+ cells) continued to express increased levels of IL-21R mRNA. On the contrary, cells cultured in the presence of IL-15 exhibited reduced levels of IL-21R mRNA (Fig. 6B).

The differences in IL-21R mRNA expression were also detected at the protein level (Fig. 7). CB/CD56+ cells, cultured with or without IL-21, expressed very low levels of IL-21R (Fig. 7C). On the contrary, freshly isolated CB/CD34+ cells did not express surface IL-21R, but exhibited a progressive increase in IL-21R expression during culture with all cytokine combinations used (i.e. FL, FL/IL-21, FL/IL-15 or FL/IL-15/IL-21, always in the presence of HC) (Fig. 7A). After day 15, IL-21R expression gradually decreased in CB/CD34+ cells cultured in the presence of IL-15, although it continued increasing in cells cultured only with FL or FL/IL-21. The decrease in IL-21R expression was inversely related to the appearance of CD56+ cells in the cultures. Gating on CD56+ cells in FACS analysis revealed that CB/CD34-developing NK cells expressed very low levels of surface IL-21R (Fig. 7B), comparable to their mature CB/CD56+ counterparts.



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  		Fig. 7. IL-21R cell-surface expression. (A) IL-21R+ CB/CD34+ cells cultured for 28 days with FL/HC and/or FL/IL-15/HC, in the presence or absence of IL-21. (B) IL-21R+ cells in total and gated, CD56– and CD56+, CB/CD34+ cells, after 28 days of culture with FL/IL-15/HC or FL/IL-15/HC/IL-21. (C) IL-21R expression on freshly isolated or cultured (28 days) CB/CD56+ and CB/CD34+ cells. Dotted line represents the isotype control. Representative results from one CB out of three samples examined are shown.

 
Effect of IL-21 on NK precursor frequency
The acquisition of IL-21R expression before induction of differentiation toward NK cells prompted us to investigate whether IL-21 increased the frequency of CB/CD34-derived NK committed precursors. To this end, we first cultured CB/CD34+ cells for 10 days with FL or FL/IL-21 and then, following FL and IL-21 withdrawal, we estimated NK precursor frequencies, after an additional 20 days culture, in the presence of IL-15 alone. LDA revealed a significant (P < 0.001) increase of NK precursor frequency in the presence of IL-21 [1:82 ± 8 (1.23% ± 0.13) versus 1:143 ± 5 (0.70% ± 0.03)].


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that IL-21 and HC act synergistically, in combination with FL and IL-15, as a potent co-stimulator of NK cell proliferation in the course of differentiation and maturation from CB cellular populations. IL-21 was shown to enhance NK cell cytotoxicity and to induce LAK activity in CB/CD34+-derived NK cells against various types of tumor targets. Our data also provide novel information on the role of IL-21 as a regulator of cytokine production by CB-derived NK cells. In particular, we found that IL-21 modulated the potential for GM-CSF, IL-10, TNF-{alpha} and IFN-{gamma} secretion by CB-derived CD56+ cells. Furthermore, we show that CB/CD34+ cells do not express IL-21R, but they do so when driven to differentiate into NK cells.

It has been reported that IL-21 is capable of inducing an accelerated maturation of NK cells from CD34+ cells in the presence of FL and IL-15 (1–6). We have recently published that the combination of IL-15 and FL, in the presence of physiological concentrations of HC, also induces the differentiation of NK cells from CD34+ cells derived from CB (19) and that HC positively affects the proliferation and cytokine secretion potential of IL-15-activated adult CD56+ cells (20). The data we present herein show the capacity of IL-21 to act synergistically with HC to significantly increase the rates of proliferation, differentiation and/or maturation in CB/CD34+-derived CD56+ cells and CB/CD56+ cells. Neither CB/CD34+ nor CB/CD56+ cells survived in the presence of IL-21 alone. Proliferation and differentiation of CB/CD34+ could not be supported in the absence of FL, since HSC do not respond to IL-15, as they do not express receptors for IL-15 (26). As expected, FL, in the presence or absence of IL-21, induced CB/CD34+ proliferation, but not differentiation into NK cells.

In addition to stimulating expansion of the CD56+ cell population, IL-21, in combination with HC, also co-stimulated effector responses, such as cytotoxicity and cytokine production. CB/CD34+-derived CD56+ cells grown in the presence of FL and IL-15, lysed K562 targets much more efficiently when IL-21 was added at culture initiation, in agreement with previous reports (1). More prominent differences were observed when testing the LAK activity of the same cells as effectors. To our knowledge, this is the first report ascribing LAK cytotoxic function to CD56+ effectors originating from CB/CD34+ cells. IL-21 did not affect NK and LAK cytotoxicity of the more mature CB/CD56+ cells.

The other major effector function of NK cells is their capacity to produce a series of immunomodulatory cytokines. As already mentioned, HC has been found to positively affect the cytokine secretion potential of adult CD56+ cells (20). The presence of IL-21 has been previously reported to enhance IFN-{gamma} production by human (32) and murine (4, 8) NK cells, activated with IL-15, IL-2 or IL-18. In agreement with previously reported data on murine NK cells (4), our results indicate that the presence of IL-21, in combination with FL, IL-15 and HC and the co-addition of IL-12 plus IL-18, significantly enhanced the production of IL-10 in CB-derived NK cells. IL-12 in synergism with IL-2 (27, 33), IL-15 (23) or IL-18 (34) has been demonstrated to induce IL-10 production by NK cells. Our data provide additional information concerning the cytokines that enhance IL-10 production by immature and mature NK cells, thus strengthening the prominent inductive effect of IL-21 on IL-10 secretion.

NK cells derived from the CB/CD34+ population in the presence of FL/IL-15/HC acquire the capacity to produce low levels of IFN-{gamma} and TNF-{alpha}, even though much less than their more mature counterparts. This ability was not enhanced by the presence of IL-21. Additionally, IL-21 could not further increase IFN-{gamma} production by CB/CD56+ cells, which in the presence of FL/IL-15/HC and the co-stimulation with IL-12/IL-18 produced high amounts of this cytokine. Goodier and Londei (30) have shown that IL-10 exerts a negative effect on IFN-{gamma} production by human mature NK cells. Considering the enhanced levels of IL-10 production by NK cells derived from cultures in the presence of IL-21, we also should have observed an IL-10-mediated decreased production of IFN-{gamma} in these cultures. The fact that this was not the case may be attributed to a masking effect caused by the co-addition of IL-18 during the last 2 days of incubation, which in synergism with IL-12 (34) or with IL-10 (35) is a potent inducer of IFN-{gamma}. If this were the case, addition of IL-12 alone, which in combination with IL-15 is also capable of inducing IFN-{gamma} production by NK cells (34), or the neutralization of the endogenously produced IL-10, would uncover the negative effect of IL-10, allowing the detection of decreased IFN-{gamma} secretion. Indeed, a 48-h incubation of the FL/IL-15/IL-21 exposed cultures with IL-12 alone or with IL-12 and IL-18 in the presence of IL-10 neutralizing antibodies, induced considerably lower levels of IFN-{gamma} production by the CD56+ cells as compared with those produced in cultures stimulated with FL/IL-15.

In the presence of IL-21, TNF-{alpha} production by CB/CD56+ cells was significantly decreased. This could not be attributed to increased levels of IL-10, which has been previously reported to negatively influence TNF-{alpha} production (28, 29), since neutralization of the endogenously produced IL-10, in the presence of IL-21, did not restore the levels of TNF-{alpha} production.

Moreover, we showed that IL-21, in combination with HC, enhanced the levels of GM-CSF produced by NK cells derived from CB populations cultured in the presence of FL/IL-15/HC and co-stimulated with IL-12/IL-18. Previous studies have reported the enhanced secretion of GM-CSF by human NK cells in the presence of IL-12, IL-18 and IL-15 (23, 34) and HC (20).

Parrish-Novak et al. (1) have previously reported that IL-21, together with FL and IL-15, promotes the differentiation of NK cells in vitro from CD34+ BM progenitors. However, in this study it was not shown if, and at which stage of NK differentiation, IL-21R was expressed. Using RT–PCR, we examined IL-21R mRNA levels in CB/CD34+ cells undergoing differentiation into CD56+ cells in the presence of FL/IL-15/HC, with or without IL-21. Although CB/CD34+ cells did not express IL-21R mRNA at culture initiation, IL-21R-specific mRNA expression was detected once differentiation was induced. IL-21R mRNA levels transiently increased above those detected in CB/CD56+ cells, but, thereafter, progressively decreased at CB/CD56+ cell levels. IL-21R expression at the protein level paralleled that of mRNA. Interestingly, high surface protein expression was detected only on CD56– cells, while the emerging CD56+ cells expressed very low levels, comparable to those expressed by their CB/CD56+ mature counterparts.

The fact that IL-21R was not detected in CB-isolated CD34+ progenitors, but was induced when these cells were driven to differentiate into NK cells, indicates that IL-21R expression might be characteristic of NK precursors, probably representing an early marker of commitment. To investigate this possibility, both IL-21R mRNA and protein levels were examined in CB/CD34+ cells cultured in the absence of IL-15, but in the presence of FL or FL/IL-21. Yu et al. (26) have previously reported that FL promotes the generation of NK cell progenitors responsive to IL-15 in BM-derived CD34+ cells. Herein, we show that FL, in the presence or absence of IL-21, induced high levels of IL-21R mRNA and cell-surface protein expression on CB/CD34+ cells, which persisted until culture termination (day 28). Furthermore, LDA revealed that IL-21 induced a significant increase in CB/CD34-NK progenitors, responsive to IL-15-induced differentiation. The biological significance of this transient high expression of IL-21R, during commitment of hematopoietic progenitors toward NK cells, remains to be elucidated.

In summary, these studies demonstrate, for the first time, that FL promotes commitment of precursor NK cells responsive to IL-21 from CB/CD34+, since the latter do not express IL-21R, but do so at a later stage of differentiation. We also show that CB/CD34+ progenitors acquire high LAK activity under the driving force of IL-21 and that IL-21, in combination with HC and IL-12/IL-18, promotes high IL-10 production by CB-derived NK cells. The finding that IL-21, apart from IL-10, modulates TNF-{alpha}, GM-CSF and IFN-{gamma} production, together with its potentiating effect on proliferation and cytotoxic activities, in the presence of physiological concentrations of HC, strengthens its prominent role in innate immunity and the immunoregulatory network, under conditions mimicking the in vivo active cortisol-containing microenvironment of blood.


   Acknowledgements
 
Supported by a grant from the Regional Operational Program Attika no. 20, MIS code 59605GR to M.P. The authors wish to thank D. Foster for kindly providing IL-21.


   Abbreviations
 
BM   bone marrow
CB   cord blood
FL   Flt3-ligand
GM-CSF   granulocyte macrophage colony-stimulating factor
HC   hydrocortisone
HSC   hematopoietic stem cell
LAK   lymphokine-activated killer
LDA   limiting dilution analysis
RT   reverse transcription
TNF   tumor necrosis factor

   Notes
 
Transmitting editor: M. Reth

Received 14 December 2004, accepted 4 October 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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