Cytokine Production and Killer Activity of NK/T-NK Cells Derived with IL-2, IL-15, or the Combination of IL-12 and IL-18 This information is current as Bernard R. Lauwerys, Nathalie Garot, Jean-Christophe of September 28, 2021. Renauld and Frédéric A. Houssiau J Immunol 2000; 165:1847-1853; ; doi: 10.4049/jimmunol.165.4.1847 http://www.jimmunol.org/content/165/4/1847 Downloaded from References This article cites 31 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/165/4/1847.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 Fre´de´ric A. Houssiau2* 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 Downloaded from 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 produc- tion 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 http://www.jimmunol.org/ 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. The Journal of Immunology, 2000, 165: 1847–1853. atural killer cells are thought to play a critical role in the have indicated that IL-15, but not IL-2, is required for NK cell defense mechanisms against pathogens and transformed ontogeny. Thus, mice genetically deficient for the IL-2R ␥-chain by guest on September 28, 2021 N cells. They display MHC-unrestricted cytotoxicity and gene, involved in IL-2 and IL-15 signaling pathways, lack NK produce various cytokines, such as IFN-␥ and TNF-␣, that further cells (16), whereas IL-2-KO mice have NK cells, although their enhance their cytotoxic effects and contribute to modulate both function is impaired (17, 18). The important role of IL-12 and innate and acquired immune responses. Thus, IFN-␥ production IL-18 as NK cell-activating factors was confirmed by the obser- by NK cells may skew immune responses toward a Th1 pattern, vation that IL-18- and IL-12-deficient mice displayed impaired NK in particular during infection with Listeria monocytogenes (1). cell activity (19). Similarly, IL-5 production by NK cells was shown to contribute We and others recently unmasked a striking synergy between to the eosinophilic reaction in a mouse model of allergic IL-12 and IL-18 for the proliferation and activation of murine NK inflammation (2). cells (20, 21). NK cells derived with the latter cytokines consisted The proliferation and activation of NK cells are controlled by of a homogeneous population, whereas those derived with IL-2 or cytokines such as IL-2 (3, 4), IL-12 (5, 6), IL-15 (7–9), and IL-18 IL-15 belonged to two different populations, namely NK and (10–15). Interestingly, observations made in knockout (KO)3 mice T-NK cells, the latter expressing both NK and T cell surface mark- ers. Interestingly, NK cells stimulated with IL-12/IL-18 secreted large amounts of IFN-␥, whereas NK cells grown in IL-2 or IL-15 *Rheumatology and †Experimental Medicine Units, Christian de Duve Institute of Cellular Pathology, Universite´catholique de Louvain, Brussels, Belgium; and ‡Lud- did not. This observation prompted us to further compare the func- wig Institute for Cancer Research, Brussels, Belgium tional characteristics of NK/T-NK cells derived in IL-2, IL-15, or Received for publication November 12, 1999. Accepted for publication May IL-12/IL-18. 31, 2000. Here, we describe 1) that murine NK and T-NK cell populations The costs of publication of this article were defrayed in part by the payment of page display distinct cytokine production profiles according to the charges. This article must therefore be hereby marked advertisement in accordance growth factors they are derived and stimulated with and 2) that with 18 U.S.C. Section 1734 solely to indicate this fact. IL-12/IL-18-derived NK cells exert a unique killer activity com- 1 This work was supported in part by grants from the Centre de Recherche Interuni- versitaire en Vaccinologie; the Fonds National de la Recherche Scientifique et Me´di- pared with NK cells derived in IL-2 or IL-15. cale; the Belgian Federal Service for Scientific, Technical, and Cultural Affairs; the Ope´ration Te´le´vie; and the Actions de Recherche Concerte´es (Communaute´Franc¸aise Materials and Methods de Belgique, Direction de la Recherche Scientifique). J.-C.R. is a Research Associate with the Fonds National de la Recherche Scientifique, Belgium. Cytokines and Abs 2 Address correspondence and reprint requests to Dr. Frederic A. Houssiau, Service Murine rIL-18 was purchased from Peprotech EC (London, U.K.). Murine de Rhumatologie UCL 5390, Universite´catholique de Louvain, Avenue Hippocrate, rIL-12 and human rIL-15 (which is active on murine cells) were obtained 10 B-1200 Brussels, Belgium. E-mail address: [email protected] from R&D Systems Europe (Abingdon, U.K.). Human rIL-2 was pur- 3 Abbreviations used in this paper: KO, knockout; FasL, Fas ligand. chased from Eurocetus (Chiron, Amsterdam, The Netherlands), and murine Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 1848 CYTOKINE PRODUCTION AND KILLER ACTIVITY OF NK/T-NK CELLS rIFN-␥ was a kind gift from Dr. W. Fiers (University of Ghent, Ghent, 51Cr release Ϫ minimal 51Cr release (targets alone)]/[maximal 51Cr release Belgium). The following cytokine concentrations were used: 1 ng/ml for (targets in 1% Triton) Ϫ minimal 51Cr release]) ϫ 100. IL-12; 100 ng/ml for IL-18; 50 ng/ml for IL-15 (except if otherwise indi- cated); 100 U/ml for IL-2; and 100 ng/ml for IFN-␥. Anti-Fas ligand (FasL) Annexin V labeling assays mAb was purchased from PharMingen (San Diego, CA) and used at a 4 concentration of 20 ␮g/ml. An Armenian hamster IgG1 purchased from Purified NK cells were seeded at 1 ϫ 10 /well in U-shaped microtiter 5 PharMingen (20 ␮g/ml) was used as isotype-matched control. plates with 1 ϫ 10 A20 cells. After 24 h, cells were tested for apoptosis by a 15-min incubation with FITC-conjugated annexin V (Genzyme Animals and cellular preparations Diagnostics, Cambridge, MA; 0.5 ␮g/ml) and propidium iodide (Sigma, 1 ␮g/ml) in HEPES buffer (10 mM HEPES-NaOH (pH 7.4), 150 mM ϫ ␥ (NZB NZW)F1 hybrids (BWF1), C57BL/6, 129, and IFN- R-KO 129 NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) before being analyzed mice were bred in our animal facility. The latter animals, initially derived by flow cytometry. by Dr. S. Huang and Dr. M. Aguet (22), were obtained through the courtesy of Dr. F. Brombacher (Max Planck Institute for Immunobiology, Freiburg, RNA extraction and RT-PCR analyses Germany). MRL/lpr mice were purchased from Harlan (Oxon, U.K.). For most experiments, we used BWF1 spleen cells that spontaneously produce Purified NK and T-NK cells were lyzed in TriPur (Boehringer Mannheim, Mann- huge amounts of Ig.