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Cytokine Production and Killer Activity of NK/T-NK Cells Derived with IL-2, IL-15, or the Combination of IL-12 and IL-18¹

Bernard R. Lauwerys^*, Nathalie Garot^*, Jean-Christophe Renauld^, and Frédéric A. Houssiau^2,*

^* Rheumatology and Experimental Medicine Units, Christian de Duve Institute of Cellular Pathology, Université catholique de Louvain, Brussels, Belgium; and Ludwig Institute for Cancer Research, Brussels, Belgium

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5⁺CD3^-), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5⁺CD3^-) and T-NK cells (DX5⁺CD3⁺). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN- and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-R knockout mice demonstrated that IFN- was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Natural killer cells are thought to play a critical role in the defense mechanisms against pathogens and transformed cells. They display MHC-unrestricted cytotoxicity and produce various cytokines, such as IFN- and TNF-, that further enhance their cytotoxic effects and contribute to modulate both innate and acquired immune responses. Thus, IFN- production by NK cells may skew immune responses toward a Th1 pattern, in particular during infection with Listeria monocytogenes (1). Similarly, IL-5 production by NK cells was shown to contribute to the eosinophilic reaction in a mouse model of allergic inflammation (2).

The proliferation and activation of NK cells are controlled by cytokines such as IL-2 (3, 4), IL-12 (5, 6), IL-15 (7, 8, 9), and IL-18 (10, 11, 12, 13, 14, 15). Interestingly, observations made in knockout (KO)³ mice have indicated that IL-15, but not IL-2, is required for NK cell ontogeny. Thus, mice genetically deficient for the IL-2R -chain gene, involved in IL-2 and IL-15 signaling pathways, lack NK cells (16), whereas IL-2-KO mice have NK cells, although their function is impaired (17, 18). The important role of IL-12 and IL-18 as NK cell-activating factors was confirmed by the observation that IL-18- and IL-12-deficient mice displayed impaired NK cell activity (19).

We and others recently unmasked a striking synergy between IL-12 and IL-18 for the proliferation and activation of murine NK cells (20, 21). NK cells derived with the latter cytokines consisted of a homogeneous population, whereas those derived with IL-2 or IL-15 belonged to two different populations, namely NK and T-NK cells, the latter expressing both NK and T cell surface markers. Interestingly, NK cells stimulated with IL-12/IL-18 secreted large amounts of IFN-, whereas NK cells grown in IL-2 or IL-15 did not. This observation prompted us to further compare the functional characteristics of NK/T-NK cells derived in IL-2, IL-15, or IL-12/IL-18.

Here, we describe 1) that murine NK and T-NK cell populations display distinct cytokine production profiles according to the growth factors they are derived and stimulated with and 2) that IL-12/IL-18-derived NK cells exert a unique killer activity compared with NK cells derived in IL-2 or IL-15.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokines and Abs

Murine rIL-18 was purchased from Peprotech EC (London, U.K.). Murine rIL-12 and human rIL-15 (which is active on murine cells) were obtained from R&D Systems Europe (Abingdon, U.K.). Human rIL-2 was purchased from Eurocetus (Chiron, Amsterdam, The Netherlands), and murine rIFN- was a kind gift from Dr. W. Fiers (University of Ghent, Ghent, Belgium). The following cytokine concentrations were used: 1 ng/ml for IL-12; 100 ng/ml for IL-18; 50 ng/ml for IL-15 (except if otherwise indicated); 100 U/ml for IL-2; and 100 ng/ml for IFN-. Anti-Fas ligand (FasL) mAb was purchased from PharMingen (San Diego, CA) and used at a concentration of 20 µg/ml. An Armenian hamster IgG1 purchased from PharMingen (20 µg/ml) was used as isotype-matched control.

Animals and cellular preparations

(NZB x NZW)F₁ hybrids (BWF₁), C57BL/6, 129, and IFN-R-KO 129 mice were bred in our animal facility. The latter animals, initially derived by Dr. S. Huang and Dr. M. Aguet (22), were obtained through the courtesy of Dr. F. Brombacher (Max Planck Institute for Immunobiology, Freiburg, Germany). MRL/lpr mice were purchased from Harlan (Oxon, U.K.). For most experiments, we used BWF₁ spleen cells that spontaneously produce huge amounts of Ig.

Splenocytes from 8-wk-old female mice were prepared by Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation. Adherent cells were removed by nylon wool filtration, and the remaining cells were cultured at a density of 3 x 10⁶ cells/well in 24-well plates in DMEM supplemented with 10% FCS, 50 µM 2-ME, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.25 mM L-glutamine, in the presence of IL-2, IL-15, or a combination of IL-12 and IL-18. After 3 days, cells were harvested and centrifuged on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient. Magnetic cell sorting was performed on the low density fraction using a mixture of anti-CD4- and anti-CD8-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), to eliminate T cells. For cells derived with IL-12/IL-18, the negatively selected CD4^-CD8^- cell fraction consisted of an homogeneous population of DX5⁺CD3^- NK cells (Fig. 1A), referred to as NK IL-12/IL-18 (between 95 and 98% DX5⁺ and <1% CD3⁺). By contrast, for cells derived with IL-2 or IL-15, the negatively selected fraction contained two distinct populations, namely DX5⁺CD3^- NK cells and DX5⁺CD3⁺ T-NK cells (Fig. 1, B and C), that were further separated by flow cytometry (FACSvantage SE, Becton Dickinson, Mountain View, CA) using a FITC-labeled anti-CD3 mAb into NK or T-NK cells, referred to as NK IL-2/T-NK IL-2 or NK IL-15/T-NK IL-15. After sorting, NK IL-2 and NK IL-15 cells were >98% DX5⁺ and < 1% CD3⁺, whereas T-NK IL-2 and T-NK IL-15 cells were >98% DX5⁺ and >98% CD3⁺. The anti-CD3 and anti-DX5 mAbs used to determine the purity of T and T-NK cell populations were purchased from PharMingen. Labeling experiments were performed in the presence of an anti-CD16/CD32 (FcRIII/II) mAb (10 µg/ml) purchased from PharMingen. Because BWF₁ mice lack NK1.1 expression, we did not use this NK cell marker.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. NK/T-NK cell populations derived from splenocyte cultures in response to IL-12 and IL-18 (A), IL-2 (B), or IL-15 (C). Nylon wool-filtered BWF1 splenocytes were supplemented with IL-2, IL-15, or IL-12 and IL-18. After a 3-day culture, cells were centrifuged on a discontinuous Percoll gradient. The low density cells were further depleted from T cells by cell sorting using a mixture of anti-CD4- and anti-CD8-coated magnetic beads. The resulting CD4-CD8- cells were double-labeled with an anti-DX5 and an anti-CD3 mAb. The upper right DX5+CD3+ population and the lower right DX5+CD3- population correspond to T-NK and NK cells, respectively.

Purified NK and T-NK cells were seeded for 24 h in triplicate or quadruplicate cultures at a density of 3 x 10⁵/well in microtiter plates in the presence of the indicated cytokine(s). IL-4 and IFN- titers were determined in the supernatants using ELISA kits purchased from BioSource Europe (Nivelles, Belgium). IL-5 and IL-13 were measured using ELISA kits supplied by Amersham Life Science (Little Chalfont, U.K.) and R&D Systems (Minneapolis, MN), respectively. Total Ig titers were determined by a standard ELISA technique using LO-MK-2 as coating Ab and peroxidase-labeled LO-MK-1 as a detecting Ab (both are anti- light chain Abs purchased from the Unit of Experimental Immunology, Université catholique de Louvain, Brussels, Belgium).

Coculture experiments

Purified NK and T-NK cells (5 x 10⁴/well) were cocultured with syngeneic splenocytes (5 x 10⁵/well) in quadruplicate microtiter wells. In other experiments, purified NK cells (1 x 10⁴/well) were added to murine A20 lymphoma cells (ATCC TIB-208; 5 x 10⁴/well). Thymidine incorporation studies (overnight pulse with 0.5 µCi [³H]thymidine/well) and cell countings after trypan blue exclusion (Seromed, Berlin, Germany) were performed after 2 days of coculture. Supernatants were harvested at day 7 for measurement of total Ig titers. For double chamber experiments, 5 x 10⁵ purified NK cells were cocultured with 3 x 10⁶ syngeneic splenocytes in 24-well plates, in either single or double chambers, using cell culture inserts (Nalge Nunc, Naperville, IL). LPS was purchased from Sigma (St. Louis, MO).

Cytotoxicity assays

Yac-1 cells (1 x 10³ cells/well), a murine NK cell target, were ⁵¹Cr labeled and incubated for 4 h in U-shaped microtiter wells with purified NK cells at various E:T ratios in quadruplicate cultures. ⁵¹Cr release was determined in supernatants, and specific lysis was calculated as the ratio: ([measured ⁵¹Cr release - minimal ⁵¹Cr release (targets alone)]/[maximal ⁵¹Cr release (targets in 1% Triton) - minimal ⁵¹Cr release]) x 100.

Annexin V labeling assays

Purified NK cells were seeded at 1 x 10⁴/well in U-shaped microtiter plates with 1 x 10⁵ A20 cells. After 24 h, cells were tested for apoptosis by a 15-min incubation with FITC-conjugated annexin V (Genzyme Diagnostics, Cambridge, MA; 0.5 µg/ml) and propidium iodide (Sigma, 1 µg/ml) in HEPES buffer (10 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂) before being analyzed by flow cytometry.

RNA extraction and RT-PCR analyses

Purified NK and T-NK cells were lyzed in TriPur (Boehringer Mannheim, Mannheim, Germany), and total RNA was purified by chloroform extraction. cDNA was synthesized by using oligo(dT) primers (Boehringer Mannheim) and murine Moloney leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD). Diluted cDNA was amplified by PCR using recombinant Taq DNA polymerase (Takara, Shiga, Japan) and specific primers for murine ß-actin (5'-ATGGATGACATATCGCTGC-3'; 3'-GTCTCCGTCTCCGTCCAC-5'), IL-4 (5'-ATGGGTCTCAACCCCCAGCTA-3'; 3'-GCATGGTGGCTCAGTACTACG-5'), IL-5 (5'-AAGGATGCTTCTGCACTTGA-3'; 3'-ACACCAAGGAACTCTTGCA-5'), IL-9 (5'-GATGATTGTACCACACCGTG-3'; 3'-CCTTTGCATCTCTGTCTTCTGG-5'), IL-10 (5'-GAGACTTGCTCTTGCACTAC-3'; 3'-CCTGGAGTCCAGCAGACTCA-5'), IL-13 (5'-ATGGCGCTCTGGGTGACTGCAG-3'; 3'-GAAGGGGCCGTGGCGAAACAGTTG-5'), IFN- (5'-GACAATCAGGCCATCAGCAAC-3'; 3'-CGCAATCACAGTCTTGGCTAA-5'), and perforin (5'-TGCTACACTGCCACTCGGTCA-3'; 3'-TTGGCTACCTTGGAGTGGGAG-5'). PCR was performed as follows: 1 min at 94°C, followed by 1 min at 63°C for IL-13, 60°C for ß-actin, 57°C for perforin, 55°C for IL-4 and IFN-, 52°C for IL-9 and IL-10, 49°C for IL-5, and by 2 min at 72°C for 20 (ß-actin and IFN-), 30 (IL-4 IL-5, IL-10, and perforin), 33 (IL-13), and 35 (IL-9) cycles. The RT-PCR products were analyzed by ethidium bromide-stained agarose gel electrophoresis. For semiquantitative perforin gene expression studies by RT-PCR analyses, serially diluted cDNA was amplified, and net fluorescence intensity of the PCR products was measured on a Kodak ImageStation 440CF (Eastman Kodak, Rochester, NY). Ratios between perforin and ß-actin counts were calculated for dilutions where amplification was linear. PCR products were sequenced with an automated fluorescence base system (Applied Biosystems 377) using cycle sequencing with standard FS dye Deoxy Terminator chemistry (Perkin-Elmer Applied Biosystems, Foster City, CA) and their homology with the Mus musculus cDNA perforin sequence (accession no. M23182) was confirmed.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Distinct cytokine production by NK/T-NK cells derived with IL-2, IL-15, or IL-12/IL-18

NK cell populations can be readily derived from murine splenocytes by addition of IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived in response to the latter cytokines consist of a homogeneous population of DX5⁺CD3^- NK cells (Fig. 1A), those derived with IL-2 or IL-15 belong to two different populations, namely DX5⁺CD3^- NK cells and DX5⁺CD3⁺ T-NK cells (Fig. 1, B and C). In a first set of experiments, we investigated whether these different T and T-NK cell populations displayed distinct patterns of cytokine production. NK and T-NK cells were purified from spleen cells stimulated with IL-2, IL-15, or IL-12/IL-18 as described in Materials and Methods and further cultured with the same cytokine(s) for 24 h. Cytokine gene expression analyses were performed by RT-PCR, and cytokine concentrations were measured by ELISA in NK or T-NK cell culture supernatants. As shown in Fig. 2, only NK cells derived in IL-12/IL-18 and T-NK cells derived in IL-2 expressed detectable amounts of cytokine messages, with the exception of IL-13 production by IL-2-stimulated NK cells. Interestingly, T-NK cells stimulated with IL-2 produced IL-4, IL-5, and IL-9 but no IFN-, whereas NK cells derived in IL-12/IL-18 produced IFN- but no IL-4, IL-5, or IL-9. Both T-NK cells cultured in IL-2 and NK cells derived in IL-12/IL-18 produced IL-10 and IL-13. NK and T-NK cells derived in IL-15 produced none of the aforementioned cytokines, even when the cytokine was added at a concentration of 500 ng/ml.

View larger version (82K): [in this window] [in a new window]   FIGURE 2. Cytokine gene expression by NK/T-NK cell populations. RT-PCR analyses were performed with primers specific for murine ß-actin, IL-4, IL-5, IL-9, IL-10, IL-13, and IFN- on cDNA synthesized from RNA extracted from purified BWF1 NK/T-NK cells derived and restimulated for 24 h with the indicated cytokines. RT-PCR products were analyzed by agarose gel electrophoresis.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Cytokine production by NK/T-NK cell populations. BWF1 IL-12/IL-18 NK cells (right), and IL-2 T-NK cells (left) were purified as described in Materials and Methods and restimulated with IL-2 () or with IL-12/IL-18 (). Cytokine concentrations (mean ± SEM) were measured by ELISA in supernatants from duplicate cultures. One arbitrary unit corresponds to 100 pg/ml for IL-4, IL-5, and IL-13 and 2000 pg/ml for IFN-. Data are representative of two distinct experiments. Similar results were observed with C57BL/6 splenocytes (data not shown). In two additional experiments, no IL-4 production was detected in supernatants from T-NK cells stimulated with as high an IL-15 concentration as 500 ng/ml (data not shown).

The observation that IL-2-stimulated T-NK cells produced type 2 cytokines such as IL-4, IL-5, and IL-9, whereas IL-12/IL-18-stimulated NK cells did not, prompted us to evaluate whether NK cell populations exerted distinct regulatory effects, in particular on spontaneous Ig production by autologous splenocytes. BWF₁ mice were used for these experiments because their splenocytes spontaneously produce large amounts of Ig. NK cell populations were purified from spleen cells stimulated with IL-2 or IL-12/IL-18, washed, and cocultured with freshly isolated syngeneic splenocytes. Supernatants were tested for Ig titers after a 7-day culture. As shown in Fig. 4, addition of NK cells derived in IL-12/IL-18 inhibited Ig production, whereas NK cells or T-NK cells derived in IL-2 did not. Rather, addition of T-NK cells grown in IL-2 up-regulated Ig production by autologous splenocytes.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Effects of NK cells on spontaneous Ig production by autologous splenocytes. Total Ig titers were measured in the supernatants of BWF1 splenocyte cultures in the presence or in the absence of autologous NK/T-NK cells derived with the indicated cytokines. Results are expressed as mean (±SEM) percent of basal Ig production measured in quadruplicate cultures. Data are representative of two distinct experiments.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Effects of IL-12/IL-18-derived NK cells on Ig production by IFN-R-KO mice. Splenocytes from wild-type (top) or IFN-R-KO (bottom) 129 mice were cultured with the indicated doses of LPS in the presence () or in the absence () of autologous NK cells derived in response to IL-12/IL-18. Ig titers were measured in the supernatants. Results are expressed as mean (±SEM) concentrations measured in triplicate cultures. Data are representative of two distinct experiments.

We hypothesized that the inhibitory effect of IL-12/IL-18-derived NK cells on Ig production was due to their cytotoxic activity against splenocytes. As indicated in Table I, addition of IL-12/IL-18 NK cells to autologous BWF₁ splenocytes increased cell mortality, as assessed by cell counting after trypan blue exclusion. By contrast, NK or T-NK cells derived in response to IL-2 or IL-15 displayed no cytotoxicity at the same E:T ratio. Similar experiments performed on Con A- and PMA-stimulated T cell blasts yielded similar results (data not shown). The distinct cytotoxic activity of IL-12/IL-18-derived NK cells, as compared with NK cells grown in IL-2 or IL-15, was further evidenced by a ⁵¹Cr release assay using Yac-1 cells as targets (Fig. 6).

View larger version (13K): [in this window] [in a new window]   FIGURE 6. Cytotoxic activity of NK cells against Yac-1 cells. 51Cr-labeled Yac-1 cells were incubated in quadruplicate with NK cells derived with the indicated cytokines. Results are expressed as mean (±SEM) percent of specific lysis at various E:T ratios.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Effects of IL-12/IL-18-derived NK cells on spontaneous Ig production in double-chamber cultures. BWF1 splenocytes were cultured in the absence or in the presence of autologous NK cells derived in IL-12/IL-18 under either single-chamber or double-chamber culture conditions. Ig titers were measured in the supernatants. Results are expressed as mean (±SEM) concentrations measured in duplicate cultures. Data are representative of two distinct experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. Effects of NK cells on A20 lymphoma cells. A20 cells were cultured for 3 days in the presence or in the absence of BWF1 NK cells derived with the indicated cytokines, at an E:T ratio of 1:5 for the proliferation assays (A) and 1:10 for the annexin V labeling assays (B). Proliferations (mean kcpm ± SEM) were measured by [3H]thymidine incorporation studies. Apoptosis was evaluated by labeling with FITC-conjugated annexin V in the presence of propidium iodide on A20 cells cultured alone (B1) or in the presence of BWF1 NK cells derived in IL-12/IL-18 (B2). Data are representative of two distinct experiments for the proliferation assays and five distinct experiments for the annexin V labeling assays.

View larger version (9K): [in this window] [in a new window]   FIGURE 9. Effects of IL-12/IL-18-derived NK cells on Ig production by MRL/lpr splenocytes. Ig production (mean ± SEM) was determined in supernatants from MRL/lpr splenocytes cultured alone or in the presence of IL-12/IL-18-derived syngeneic NK cells. Data are representative of two experiments.

View larger version (12K): [in this window] [in a new window]   FIGURE 10. Perforin gene expression in NK cells. RNA was extracted from purified NK cells derived with the indicated cytokines and cDNA was synthesized as indicated in Materials and Methods. Serially diluted cDNA was amplified using specific primers for ß-actin and perforin. Results are expressed as mean (±SEM) arbitrary units calculated as the ratio between luminescence intensity of perforin and ß-actin PCR products where amplification was linear. The data of two experiments were pooled.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although numerous recent studies, including our own, have identified the critical role of IL-18 (alone or in synergy with IL-12 or IL-10) as a potent IFN- inducer in NK cells (10, 12, 15, 20), the data available regarding other cytokines produced by NK cells are more difficult to interpret. The cytokine expression profile of NK cell populations is indeed influenced not only by the growth factor(s) used to expand and to stimulate them but also by the level of purity of the NK preparations. The latter issue is of the utmost importance for NK blasts derived in response to IL-2 or IL-15, because stimulation by either of these two cytokines generates two different cellular populations bearing NK markers, namely DX5⁺CD3^- NK cells and DX5⁺CD3⁺ T-NK cells (21), which may both contribute to cytokine production. In this respect, our observation that T-NK cells derived in response to IL-2 (and not NK cells derived and tested under the same experimental conditions) produce type 2 cytokines, such as IL-4, IL-5, and IL-9, suggests that T-NK cells can provide some help to humoral immune responses. Accordingly, we noted that addition of T-NK cells derived in IL-2 to autologous spleen cells augmented their Ig production (Fig. 4), likely via induction of type 2 cytokines. By contrast, and well in line with previous findings, we confirmed that NK cells derived in IL-12/IL-18 produce IFN- but no IL-4, IL-5 or IL-9. It should be stressed, however, that we did not observe a strict type 1/type 2 cytokine dichotomy between T-NK cells derived in IL-2 on the one hand and NK cells grown in IL-12/IL-18 on the other hand, because both subsets produced IL-10 and IL-13, two other purported type 2 cytokines.

Although IL-15 strongly stimulated the growth of NK and T-NK cells from bulk spleen cell cultures, we could not detect any cytokine production in NK/T-NK cells derived in IL-15, even added at a concentration of 500 ng/ml. The striking difference between IL-2 and IL-15 with respect to cytokine induction in T-NK cells is puzzling because IL-2 and IL-15 are purported to share the same signaling pathway through the ß- and -chains of the IL-2R (26). Our observation that IL-2 and IL-15 have distinct effects on cytokine expression by T-NK cells suggests that their signaling pathways might differ at least in certain cell types. On stimulation with an anti-CD3 mAb, T-NK cells produced large amounts of IL-4 and IFN-, irrespective of the cytokines (IL-2 or IL-15) they were derived with. A functional difference between IL-15 and IL-2 was also recently reported for cytokine production by TCRß-positive intestinal intraepithelial lymphocytes which were shown to produce IFN- after IL-2, but not IL-15, stimulation (27).

Switch-culture experiments indicated that cytokine production by NK/T-NK cells was not strictly committed by their initial culture condition. Thus, IFN-- and IL-13-producing NK cells derived in IL-12/IL-18 down-regulated their IFN- and IL-13 production when pulsed in IL-2. Conversely, type 2 cytokine-producing T/NK cells grown in IL-2 and further cultured in IL-12/IL-18 expressed IFN-, augmented their IL-13 production and down-regulated IL-4 and IL-5 expression. These results confirm that cytokine production by NK/T-NK cells is a reversible process depending on the cytokine(s) with which they are stimulated and is not associated with a fixed phenotype acquired during ontogeny.

Our results demonstrate for the first time that murine NK cells derived in response to IL-12 and IL-18 are much more cytotoxic effectors against syngeneic and tumoral targets than NK cells grown in IL-2 or IL-15. Interestingly, the cytotoxicity of NK cells derived in IL-2 or IL-15 was not enhanced to the level observed with IL-12/IL-18 NK cells by a 72-h pulse with IL-12/IL-18 (data not shown), while their IFN- and IL-13 productions were up-regulated as indicated above. Given the potent synergy between IL-12 and IL-18 for IFN- production by different lymphocyte subsets, including NK cells (21, 28, 29, 30), we first investigated whether the latter cytokine was responsible for the potent killer activity of NK cells derived with IL-12 and IL-18. Experiments performed on IFN-R-KO-splenocytes convincingly ruled out this hypothesis. Double-chamber experiments, by indicating that cell-to-cell contact was required for the cytotoxicity of NK cells to occur, suggested that the effect of NK cells derived in IL-12 and IL-18 was to induce target apoptosis, a hypothesis that was confimed by annexin V labeling assays performed on murine A20 lymphoma cells.

The mechanisms explaining the different level of proapoptotic activity between distinct NK cell populations according to the cytokine(s) they were derived with, remain unclear. Thus, Tsutsui et al. (11) observed that IL-18 up-regulated FasL expression on liver-derived murine NK cell clones and increased their Fas/FasL-mediated cytotoxicity. Our observations that addition of anti-FasL Ab did not inhibit the cytotoxicity of IL-12/IL-18 NK cells and that MRL/lpr NK cells derived in response to IL-12 and IL-18 inhibited Ig production by autologous spleen cells indicated that Fas/FasL interaction was not required for the cytotoxicity of IL-12/IL-18 NK cells to occur.

The effects of IL-12 and IL-18 on NK cell expression of other molecules involved in cytotoxicity have recently been studied by Hyodo et al. (31). They found that IL-12, but not IL-18, enhances mRNA expression of perforin and granzyme B in murine NK cells. Moreover, splenocytes from perforin-deficient mice incubated for 24 h with IL-12 and IL-18 failed to display NK activity, thereby demonstrating that perforin is required for the cytotoxicity of IL-12/IL-18-stimulated spleen cells. Using semiquantitative RT-PCR analyses, we observed an increased expression of the perforin gene in NK cells derived in response to IL-12/IL-18, compared with NK cells grown in IL-2 or IL15. These results, although they do not address, strictly speaking, the mechanisms underlying the unique lytic activity of IL-12/IL-18-derived NK cells, lend support to the possibility that induction of the perforin lytic pathway accounts for their preferential cytotoxicity.

Taken together, our results indicate 1) that very potent cytotoxic NK cells can be derived in response to IL-12 and IL-18, compared with IL-2 or IL-15, and 2) that type 2 cytokines are produced by IL-2-derived T-NK cells, which thereby might contribute to modulate humoral immune responses.

	Acknowledgments

 
We thank A. Tonon for running the cell sorting experiments.

	Footnotes

 
¹ This work was supported in part by grants from the Centre de Recherche Interuniversitaire en Vaccinologie; the Fonds National de la Recherche Scientifique et Médicale; the Belgian Federal Service for Scientific, Technical, and Cultural Affairs; the Opération Télévie; and the Actions de Recherche Concertées (Communauté Française de Belgique, Direction de la Recherche Scientifique). J.-C.R. is a Research Associate with the Fonds National de la Recherche Scientifique, Belgium.

² Address correspondence and reprint requests to Dr. Frederic A. Houssiau, Service de Rhumatologie UCL 5390, Université catholique de Louvain, Avenue Hippocrate, 10 B-1200 Brussels, Belgium.

³ Abbreviations used in this paper: KO, knockout; FasL, Fas ligand.

Received for publication November 12, 1999. Accepted for publication May 31, 2000.

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