Soluble Interleukin-15 Receptor α (IL-15Rα)-sushi as a Selective and Potent Agonist of IL-15 Action through IL-15Rβ/γ

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15Rα chain corresponding to the entire extracellular domain of IL-15Rα behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15Rα, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15Rβ/γ heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15Rα/β/γ membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15Rα-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15Rα-sushi. After binding to IL-15Rβ/γ, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets. Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15Rα chain corresponding to the entire extracellular domain of IL-15Rα behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15Rα, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15Rβ/γ heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15Rα/β/γ membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15Rα-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15Rα-sushi. After binding to IL-15Rβ/γ, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets. IL-15 3The abbreviations used are: IL-15interleukin-15IL-15RαIL-15R α-chainsIL-15Rα-sushisoluble IL-15Rα sushi domainILR RLIIL-15-sIL-15Rα-sushi fusion proteinSPRsurface plasmon resonancerILrecombinant human IL-15mILmouse interleukinNKnatural killer cells. is a cytokine that was originally described, like IL-2, as a T cell growth factor (1Grabstein K.H. Eisenman J. Shanebeck K. Rauch C. Srinivasan S. Fung V. Beers C. Richardson J. Schoenborn M.A. Ahdieh M. Johnson L. Alderson M.R. Watson J.D. Anderson D.M. Giri J.G. Science. 1994; 264: 965-968Crossref PubMed Scopus (1343) Google Scholar). Both cytokines belong to the four α-helix bundle family, and their membrane receptors share two subunits (the IL-2R/IL-15R β and γ chains) responsible for signal transduction (2Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Crossref PubMed Scopus (970) Google Scholar). The IL-2Rβ/γ complex is an intermediate affinity receptor for both cytokines that is expressed by most NK cells and can be activated in vitro by nanomolar concentrations of IL-2 or IL-15. The high affinity IL-2 and IL-15 receptors, such as those expressed on activated T cells, can be activated with picomolar concentrations of either cytokine, and additionally contain their own private α chain (IL-2Rα and IL-15Rα) that confers cytokine specificity and enhances the affinity of cytokine binding (3Anderson D.M. Kumaki S. Ahdieh M. Bertles J. Tometsko M. Loomis A. Giri J. Copeland N.G. Gilbert D.J. Jenkins N.A. Valentine V. Shapiro D.N. Morris S.W. Park L.S. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). interleukin-15 IL-15R α-chain soluble IL-15Rα sushi domain IL-15-sIL-15Rα-sushi fusion protein surface plasmon resonance recombinant human IL-15 mouse interleukin natural killer cells. Both cytokines play pivotal roles in innate and adaptative immunity. Whereas initial in vitro experiments have shown a large functional overlap in the effects of the two cytokines (induction of the proliferation and cytotoxicity of activated lymphocytes and NK cells, co-stimulation of B cell proliferation and immunoglobulin synthesis, and chemoattraction of T cells) (1Grabstein K.H. Eisenman J. Shanebeck K. Rauch C. Srinivasan S. Fung V. Beers C. Richardson J. Schoenborn M.A. Ahdieh M. Johnson L. Alderson M.R. Watson J.D. Anderson D.M. Giri J.G. Science. 1994; 264: 965-968Crossref PubMed Scopus (1343) Google Scholar, 4Burton J.D. Bamford R.N. Peters C. Grant A.J. Kurys G. Goldman C.K. Brennan J. Roessler E. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4935-4939Crossref PubMed Scopus (343) Google Scholar, 5Carson W.E. Giri J.G. Lindemann M.J. Linett M.L. Ahdieh M. Paxton R. Anderson D. Eisenmann J. Grabstein K. Caligiuri M.A. J. Exp. Med. 1994; 180: 1395-1403Crossref PubMed Scopus (977) Google Scholar, 6Wilkinson P.C. Liew F.Y. J. Exp. Med. 1995; 181: 1255-1259Crossref PubMed Scopus (220) Google Scholar), more recent experiments have indicated that they can exert complementary or even contrasting actions in vivo. Whereas IL-2 or IL-2Rα knock-out in mice was associated with autoimmune phenotypes with increased populations of activated T and B cells, IL-15 and IL-15Rα knock-out resulted in specific defects in NK, NK-T, intraepithelial lymphocytes, and memory CD8 T cells (7Kennedy M.K. Glaccum M. Brown S.N. Butz E.A. Viney J.L. Embers M. Matsuki N. Charrier K. Sedger L. Willis C.R. Brasel K. Morrissey P.J. Stocking K. Schuh J.C. Joyce S. Peschon J.J. J. Exp. Med. 2000; 191: 771-780Crossref PubMed Scopus (1356) Google Scholar, 8Lodolce J.P. Burkett P.R. Boone D.L. Chien M. Ma A. J. Exp. Med. 2001; 194: 1187-1194Crossref PubMed Scopus (155) Google Scholar). Furthermore, IL-2 promotes peripheral tolerance by inducing activation-induced cell death, whereas IL-15 inhibits IL-2-mediated activation-induced cell death (9Marks-Konczalik J. Dubois S. Losi J.M. Sabzevari H. Yamada N. Feigenbaum L. Waldmann T.A. Tagaya Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11445-11450Crossref PubMed Scopus (362) Google Scholar), and, unlike IL-2, IL-15 is a survival factor for CD8 memory T cells (10Ku C.C. Murakami M. Sakamoto A. Kappler J. Marrack P. Science. 2000; 288: 675-678Crossref PubMed Scopus (720) Google Scholar). In line with these observations, it has been suggested that the major role of IL-2 is to limit continuous expansion of activated T cells, whereas IL-15 is critical for initiation of T cell division and survival of memory T cells (11Li X.C. Demirci G. Ferrari-Lacraz S. Groves C. Coyle A. Malek T.R. Strom T.B. Nat. Med. 2001; 7: 114-118Crossref PubMed Scopus (265) Google Scholar). A novel mechanism of IL-15 action described recently is that of transpresentation in which IL-15 and IL-15Rα are coordinately expressed by antigen-presenting cells (monocytes and dendritic cells), and IL-15 bound to IL-15Rα is presented in trans to neighboring NK or CD8 T cells expressing only the IL-15Rβ/γ receptor (12Dubois S. Mariner J. Waldmann T.A. Tagaya Y. Immunity. 2002; 17: 537-547Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar). As a co-stimulatory event occurring at the immunological synapse, IL-15 transpresentation now appears to be a dominant mechanism for IL-15 action in vivo (13Burkett P.R. Koka R. Chien M. Chai S. Chan F. Ma A. Boone D.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4724-4729Crossref PubMed Scopus (130) Google Scholar, 14Schluns K.S. Nowak E.C. Cabrera-Hernandez A. Puddington L. Lefrancois L. Aguila H.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5616-5621Crossref PubMed Scopus (134) Google Scholar) and appears to play a major role in tumor immunosurveillance (15Kobayashi H. Dubois S. Sato N. Sabzevari H. Sakai Y. Waldmann T.A. Tagaya Y. Blood. 2005; 105: 721-727Crossref PubMed Scopus (212) Google Scholar). The IL-15Rα and IL-2Rα subunits form a sub-family of cytokine receptors in that they comprise extracellular parts, so called "sushi" structural domains (one in IL-15Rα and two in IL-2Rα), at their N terminus, which are also found in complement or adhesion molecules (16Norman D.G. Barlow P.N. Baron M. Day A.J. Sim R.B. Campbell I.D. J. Mol. Biol. 1991; 219: 717-725Crossref PubMed Scopus (207) Google Scholar). In both cases, these sushi domains have been shown to bear most of the structural elements responsible for cytokine binding. Whereas IL-2Rα alone is a low affinity receptor for IL-2 (Kd = 10 nm), IL-15Rα binds IL-15 with high affinity (Kd = 100 pm). Shedding of IL-2Rα by proteolysis is a natural mechanism that participates in the down-regulation of lymphocyte activation. IL-2Rα is cleaved by Der p1, a major mite allergen, thereby inhibiting Th1 cells and favoring an allergic environment (17Schulz O. Sewell H.F. Shakib F. J. Exp. Med. 1998; 187: 271-275Crossref PubMed Scopus (233) Google Scholar). IL-2Rα cleavage by tumor-derived metalloproteinases also results in the suppression of the proliferation of cancer-encountered T cells (18Sheu B.C. Hsu S.M. Ho H.N. Lien H.C. Huang S.C. Lin R.H. Cancer Res. 2001; 61: 237-242PubMed Google Scholar). The soluble IL-2Rα thus generated is a competitive inhibitor of IL-2 action in vitro. However, it remains a low affinity IL-2 binder and is not likely to efficiently participate in down-regulation of IL-2 activity in vivo. We have recently shown that a soluble form of the human IL-15Rα can also be naturally released from IL-15Rα-positive cells by a shedding process involving matrix metalloproteinases (19Mortier E. Bernard J. Plet A. Jacques Y. J. Immunol. 2004; 173: 1681-1688Crossref PubMed Scopus (117) Google Scholar). In contrast to soluble IL-2Rα, this soluble IL-15Rα receptor was able to bind IL-15 with high affinity and efficiently blocked proliferation driven through the high affinity IL-15Rα/β/γ signaling complex. This result was consistent with the concept of sIL-15Rα behaving, like its homolog sIL-2Rα, as an antagonist, and with the inhibitory effects of mouse sIL-15Rα in vitro or in vivo (20Ruchatz H. Leung B.P. Wei X.Q. McInnes I.B. Liew F.Y. J. Immunol. 1998; 160: 5654-5660PubMed Google Scholar, 21Smith X.G. Bolton E.M. Ruchatz H. Wei X. Liew F.Y. Bradley J.A. J. Immunol. 2000; 165: 3444-3450Crossref PubMed Scopus (79) Google Scholar). Here, we show that a soluble receptor consisting of the N-terminal structural domain of IL-15Rα (sushi domain) has the opposite action; it is able to enhance the binding as well as the bioactivity of IL-15 through the IL-15Rβ/γ intermediate affinity receptor, without affecting binding and bioactivity through the high affinity receptor. In addition, we describe fusion proteins consisting of human IL-15 and human IL-15Rα fused by flexible linkers that behave as potent superagonists of the IL-15Rβ/γ complex. Cell Culture and Cytokines—Recombinant human IL-15 (rIL-15) was purchased from Peprotech Inc. (Rocky Hill, NJ). The Mo-7e myeloid leukemia (22Meazza R. Basso S. Gaggero A. Detotero D. Trentin L. Pereno R. Azzarone B. Ferrini S. Int. J. Cancer. 1998; 78: 189-195Crossref PubMed Scopus (34) Google Scholar) and TF-1 erythroleukemia human cell lines (ATCC CRL-2003) (kindly provided by Dr. Bruno Azzarone, Institut Gustave Roussy, Villejuif, France) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 2 mm glutamine, and 1 ng/ml granulocyte macrophage-colony stimulating factor (R&D Systems, Abington, UK). TF1-β cells (23Farner N.L. Gan J. de Jong J.L. Leary T.P. Fenske T.S. Buckley P. Dunlap S. Sondel P.M. Cytokine. 1997; 9: 316-327Crossref PubMed Scopus (13) Google Scholar) were cultured in the same medium supplemented with 250 μg/ml Geneticin. The Kit 225 human T lymphoma cell line (24Hori T. Uchiyama T. Tsudo M. Umadome H. Ohno H. Fukuhara S. Kita K. Uchino H. Blood. 1987; 70: 1069-1072Crossref PubMed Google Scholar) (obtained from Dr. Doreen Cantrell, University of Dundee, UK) was cultured in RPMI 1640 medium containing 6% fetal calf serum, 2 mm glutamine, and 10 ng/ml rIL-2 (Chiron, Emeryville, CA). 32Dβ cells (25Nakamura Y. Russell S.M. Mess S.A. Friedmann M. Erdos M. Francois C. Jacques Y. Adelstein S. Leonard W.J. Nature. 1994; 369: 330-333Crossref PubMed Scopus (286) Google Scholar) (kindly provided by Dr. Warren Leonard, Bethesda, MD) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 2 mm glutamine, 0.4 ng/ml mIL-3 (R&D Systems), 10 μm 2-mercaptoethanol, 250 μg/ml Geneticin. sIL-15Rα·IL-2, sIL-15Rα-sushi·IL-2, and sIL-15Rα-sushi—sIL-15Rα·IL-2 was expressed in Chinese hamster ovary cells and prepared as described (26Bernard J. Harb C. Mortier E. Quemener A. Meloen R.H. Vermot-Desroches C. Wijdeness J. van Dijken P. Grotzinger J. Slootstra J.W. Plet A. Jacques Y. J. Biol. Chem. 2004; 279: 24313-24322Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). A similar construction was made in which the sushi domain of IL-15Rα (amino acids 1-66 of mature coding sequence) was linked to a molecule of human IL-2 (sIL-15Rα-sushi·IL-2). The sushi domain of IL-15Rα was amplified by PCR. PCR products were purified, digested with BamHI and HindIII (Fermentas, Vilnius, Lithuania) and ligated into a pQE30 expression vector. Expression was performed in Escherichia coli SG13009 cells under isopropyl 1-thio-β-d-galactopyranoside induction. After cell lysis, inclusions bodies were washed, solubilized in 6 mm guanidine HCl, 20 mm sodium phosphate, pH 7.4, 20 mm imidazole, 150 mm sodium chloride, and 1 mm dithiothreitol. The IL-15Rα-sushi was trapped in an nickel-nitrilotriacetic acid-agarose column (Qiagen) equilibrated with the solubilization buffer plus 1 mm reduced glutathione and 0.2 mm oxidized glutathione. It was then refolded via a gradient from 6 to 0 m guanidine HCl in the column buffer (27Matsumoto M. Misawa S. Tsumoto K. Kumagai I. Hayashi H. Kobayashi Y. Protein Expr. Purif. 2003; 31: 64-71Crossref PubMed Scopus (22) Google Scholar) and eluted with 250 mm imidazole. RLI and ILR Fusion Proteins—The constructions of the fusion proteins are shown in Fig. 2E. The human IL-15Rα-sushi domain (amino acids 1-77) and human IL-15 were separated by linker 20 (SGGSGGGGSGGGSGGGGSLQ) for RLI or by linker 26 (SGGGSGGGGSGGGGSGGGGSGGGSLQ) for ILR. A sequence coding for the FLAG epitope and Factor Xa binding site (DYKDDDDKIEGR) was added between the signal peptide and the coding sequences. The endogenous signal peptide of human IL-15Rα was used for RLI and the signal peptide of bovine preprolactin for ILR. These constructions were inserted between the BamHI and the Hind III sites of a pFastBac 1 (Invitrogen) expression vector thus generating two expression vectors, which were recombined in baculovirus DNA using the Bac to Bac expression system (Invitrogen). The recombinant baculoviruses were used to infect SF9 cells, and fusion proteins were expressed in the SF 900 II medium and harvested 4 days post infection. The concentrations of the fusion proteins were measured by enzyme-linked immunosorbent assay with the anti-IL-15 monoclonal antibody 247 (R & D Systems) as the capture antibody and the anti-FLAG M2-peroxydase conjugate (Sigma) as the revealing antibody. Surface Plasmon Resonance Studies—The SPR experiments were performed with a BIAcore 2000 biosensor (BIAcore, Uppsala, Sweden). rIL-15 was covalently linked to CM5 sensor chips, and the binding of increasing concentrations of sIL-15Rα·IL-2, sIL-15Rα-sushi·IL-2, or sIL-15Rα-sushi was monitored. Analysis of sensorgrams was performed using BIAlogue kinetics evaluation software. Proliferation Assays—The proliferative responses of Mo-7e, TF-1β, and Kit 225 cells to rIL-15, rIL-2, RLI, or ILR were measured by [3H]thymidine incorporation as described (19Mortier E. Bernard J. Plet A. Jacques Y. J. Immunol. 2004; 173: 1681-1688Crossref PubMed Scopus (117) Google Scholar) after 4 h in cytokine-deprived medium, 48 h culture, and 16 h with [3H]thymidine. Apoptosis—The annexin V assay was performed using a FACScan flow-cytometer and the Annexin V-FITC Apoptosis detection kit (BD Biosciences). After cytokine starvation, cells were seeded in multiwell plates at 5 × 105 cells/well in 1 ml and cultured in medium supplemented with the various reactants (rIL-15, sIL-15Rα-sushi, and RLI fusion protein). Data were acquired and analyzed using CellQuest software. Binding Assays and Internalization—Labeling with [125I]iodine of human rIL-15, sIL-15Rα-sushi, and RLI fusion protein, as well as subsequent binding experiments, were performed as described previously (19Mortier E. Bernard J. Plet A. Jacques Y. J. Immunol. 2004; 173: 1681-1688Crossref PubMed Scopus (117) Google Scholar). For internalization, cells were equilibrated at 4 °C with labeled sIL-15Rα-sushi or RLI, and the temperature was switched to 37 °C. At different time intervals, two samples were washed and centrifuged. One of the cell pellets was treated with glycine-HCl buffer (0.2 m, pH 2.5), whereas the other was treated with phosphate-buffered saline (pH 7.4) at 4 °C for 5 min. After centrifugation, total ligand binding was determined from the pellet of the cells treated with PBS, whereas the membrane-bound and internalized fractions were determined, respectively, from the supernatant and pellet of cells treated with acid pH. IL-15Rα Binding to IL-15 Is Mainly Due to the Sushi Domain—In a previous study (28Dubois S. Magrangeas F. Lehours P. Raher S. Bernard J. Boisteau O. Leroy S. Minvielle S. Godard A. Jacques Y. J. Biol. Chem. 1999; 274: 26978-26984Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), we have shown that removal of the sushi domain encoded by exon 2 of IL-15Rα resulted in a complete abrogation of IL-15 binding to membrane anchored IL-15Rα, suggesting that the sushi domain was indispensable for binding. To directly measure the contribution of the sushi domain to IL-15 binding, soluble forms of IL-15Rα containing the entire extracellular domain or only the N-terminal sushi domain were prepared and assayed for IL-15 binding in a competition assay and using SPR technology. As shown in Fig. 1A, a sIL-15Rα·IL-2 fusion protein produced in Chinese hamster ovary cells and consisting of the entire IL-15Rα extracellular domain linked to a molecule of human IL-2 (used as a tag for purification) bound IL-15 with high affinity (kon = 3.7 × 105 m-1 s-1; koff = 1.4 × 10-5 s-1; Kd = 38 pm). A similar construction linking the sushi domain of IL-15Rα to human IL-2 also bound IL-15 (Fig. 1B) but with a 10-fold lower affinity, mainly due to a more rapid off rate (kon = 3.1 × 105 m-1 s-1; koff = 1.3 × 10-4 s-1; Kd = 420 pm). A soluble sushi domain was also produced in E. coli. This sIL-15Rα-sushi also bound IL-15 with a lower affinity (kon = 2.5 × 105 m-1 s-1; koff = 3.8 × 10-4 s-1; Kd = 1.5 nm) (Fig. 1C). These results indicate that the sushi domain is responsible for most of the binding affinity of IL-15 but that it does not fully reconstitute the high affinity binding displayed by the full-length extracellular domain. Soluble IL-15Rα Proteins Inhibit IL-15 Binding to Membrane-anchored IL-15Rα—The three soluble forms of IL-15Rα were tested for their ability to compete with radioiodinated IL-15 for binding to IL-15Rα expressed by the human cell line TF-1 that also expresses the IL-15Rγ chain but not the IL-15Rβ chain (Fig. 1D). The three proteins completely inhibited IL-15 binding to TF-1 cells with IC50 values that were similar to the Kd values measured by the SPR technology: 100 pm (sIL-15Rα·IL-2), 270 pm (sIL-15Rα-sushi·IL-2), and 1.3 nm (sIL-15Rα-sushi). sIL-15Rα-sushi Increases IL-15-driven Cell Proliferation through the IL-15Rβ/γ Complex—Because the soluble sushi domain was easily produced in E. coli in high yields, it was selected for all further studies. It was first tested on cell lines that only express the IL-15Rβ/γ complex (the human Mo-7e cell line and the mouse 32Dβ cell line that expresses endogenous mouse IL-15Rγ chain and transfected human IL-15Rβ chain). As expected, the Mo-7e cell line proliferated in response to nanomolar concentrations of rIL-15 or rIL-2 (Fig. 2, A and B). Unexpectedly, addition of a fixed concentration of sIL-15Rα-sushi (10 nm)to the assay increased the proliferative response to lower concentrations of rIL-15 by ∼4-fold. By itself, sIL-15Rα-sushi did not induce any proliferative response (data not shown). Similar results were obtained for 32Dβ with a shift of ∼10-fold (data not shown). The specificity was assessed by the fact that sIL-15Rα-sushi did not affect the rIL-2-driven proliferation of Mo-7e cells (Fig. 2B). Fig. 2C shows that sIL-15Rα-sushi dose dependently (with an IC50 of 3.5 nm, similar to its Kd for IL-15) potentiated the effect of a fixed concentration of rIL-15 (1 nm) that alone induces only a low level of proliferation. RLI and ILR Fusion Proteins Are Potent Inducers of Cell Proliferation through the IL-15Rβ/γ Complex—To evaluate whether the synergistic effect of sushi on IL-15 bioactivity could be transferred by a single molecule, molecular constructs encoding fusion proteins linking IL-15 and the sushi domain were elaborated. For the two constructions, a flexible linker was introduced between the C terminus of IL-15 and the N terminus of the sushi domain (ILR) or vice versa (RLI) (Fig. 2E). Molecular models illustrating the structures of these proteins are shown in Fig. 2F. These two fusion proteins were tested for their effect on the proliferation of Mo-7e cells. As shown in Fig. 2D, both proteins induced dose-dependent induction of Mo-7e cell proliferation, with EC50s that were similar (∼25 pm) and far lower than those of rIL-15 alone (3 nm), or of an equimolar mixture of rIL-15 plus sIL-15Rα-sushi (0.9 nm). These results further confirm the synergistic effect of sIL-15Rα-sushi on the actions of IL-15 and indicate that stabilizing the IL-15·sIL-15Rα-sushi complex with a covalent linker markedly enhances this synergistic action. sIL-15Rα-sushi Increases IL-15-induced Prevention of Apoptosis, and RLI Efficiently Prevents Cellular Apoptosis—Following cytokine withdrawal, the fraction of apoptotic Mo-7e cells increased from 10 to 80% in 48 h (Fig. 3, A (panel a) and A (panel b)). When added at time zero, rIL-15 (5 nm) reduced this apoptosis to 70% (Fig. 3A, panel c). Alone, sIL-15Rα-sushi (10 nm) had no effect (Fig. 3A, panel b). However, it markedly potentiated the anti-apoptotic effect of rIL-15 (35% apoptosis at 48 h, Fig. 3A, panel c). The synergistic effect of sIL-15Rα-sushi on IL-15-mediated prevention of apoptosis was confirmed by a kinetic analysis (Fig. 3B) as well as by dose-response curves (Fig. 3C). rIL-15 acted with an IC50 of ∼1.5 nm, a value in agreement with the saturation of IL-15β/γ receptors. This IC50 was ∼10-fold lower (170 pm) in the presence of 10 nm sIL-15Rα-sushi. The RLI fusion protein markedly prevented apoptosis (Fig. 3B). On a molar basis, it was even more active than the IL-15·sIL-15Rα-sushi association, with an IC50 of ∼40 pm (Fig. 3C). sIL-15Rα-sushi Increases IL-15 Binding to Mo-7e Cells, and the RLI Fusion Protein Binds to and Is Internalized by Mo-7e Cells—As expected, Mo-7e cells bound IL-15 with intermediate affinity (Kd = 13.5 nm), with a maximal binding capacity of 800 sites/cell (Fig. 4A). The addition of sIL-15Rα-sushi (10 nm) increased the affinity of IL-15 binding (Kd = 7 nm) without significantly affecting the maximal binding capacity (1180 sites/cell). When using a radioiodinated RLI fusion protein (Fig. 4B), we detected binding to a similar number of receptor sites (730 sites/cell), and the affinity of binding (Kd = 780 pm) was markedly higher than that of IL-15. Fig. 4C shows that RLI can be efficiently and rapidly internalized. The fraction of cell-bound radioactivity decreased within ∼20 min and was accompanied by a concomitant increase in intracellular radioactivity. sIL-15Rα-sushi Does Not Affect IL-15-driven Cell Proliferation nor Inhibition of Apoptosis through the High Affinity IL-15Rα/β/γ Complex—The human lymphoma cell line Kit 225 expresses endogenous IL-15Rα, -β, and -γ chains, and the human TF-1β cell line expresses endogenous IL-15Rα and -γ chains plus the transfected human IL-15Rβ chain. Consequently, these cell lines proliferate in response to low, picomolar concentrations of IL-15, as shown in Fig. 5 (A and B) (EC50 = 19 pm and 21 pm, respectively). In contrast to what was found using Mo-7e or 32Dβ cells, addition of equimolar concentrations of sIL-15Rα-sushi to IL-15 did not significantly affected the IL-15 dose-response curve on either cell type. The ILR fusion protein was as active as rIL-15 on the two cell lines. The RLI was also as active as rIL-15 on Kit 225 cells but was ∼16-fold more efficient (EC50 = 1.2 pm) than rIL-15 on TF-1β cells. The effects of sIL-15Rα-sushi and RLI were further analyzed on TF-1β cell apoptosis induced by cytokine deprivation. Histograms showing these effects are given in Fig. 5C, whereas the kinetics and dose responses of these effects are shown in Fig. 5, D and E, respectively. rIL-15 dose and time dependently inhibited TF-1β apoptosis. sIL-15Rα-sushi alone had no effect and did not change the effect of IL-15 IC50 = 6.5 pm for rIL-15 or sIL-15Rα-sushi plus rIL-15. The ILR fusion protein was as active as rIL-15, whereas RLI was about three times more active (IC50 = 2.5 pm). IL-15, sIL-15Rα-sushi, and RLI Binding to TF-1β Cells—Insofar as IL-15Rα-sushi did not affect IL-15 proliferation of TF-1β, we examined its effect on IL-15 binding over a wide concentration range (Fig. 6A). Scatchard analysis of the saturation binding curve indicated the presence of two classes of IL-15 binding sites, compatible with the presence of a small number of high affinity binding sites (IL-15Rα/β/γ complexes, Kd = 22 pm, Bmax = 100 sites/cell) plus higher amounts of intermediate affinity binding sites (IL-15Rβ/γ complexes, Kd = 30 nm, 2800 sites/cell). sIL-15Rα-sushi induced an increase in IL-15 binding that, according to Scatchard analysis, was mainly due to an increase in the affinity of IL-15 binding for the intermediate-affinity component (Kd = 3.5 nm). To more specifically test the effect of sIL-15Rα on the high affinity component, its effect was analyzed on the binding of a low concentration of radiolabeled IL-15 (200 pm) that mainly targets the high affinity receptor. As shown in Fig. 6B, sIL-15Rα-sushi, at concentrations of up to 25 nm, did not affect this binding. The binding of radiolabeled sIL-15Rα-sushi to TF-1β cells (Fig. 6C) revealed a specific binding component that was strictly dependent on the presence of rIL-15. In the presence of 1 nm rIL-15, the Kd reflecting sIL-15Rα-sushi binding was 3.5 nm, a value compatible with its affinity for IL-15, with a maximal binding capacity (3300 sites/cell) compatible with the number of IL-15 intermediate binding sites. As further shown in Fig. 6D, the radiolabeled sIL-15Rα-sushi was efficiently internalized. Radiolabeled RLI fusion protein also bound to TF-1β cells (Fig. 6E). A single specific binding component was observed with a Kd of 250 pm and a maximal capacity (4000 sites/cell) again comparable to the number of IL-15 intermediate affinity binding sites. Once bound, the RLI was also efficiently internalized (Fig. 6F). Deletion of the exon 2-encoded sushi domain of human IL-15Rα was formerly shown to completely abrogate IL-15 binding, indicating the dispensable role of the sushi domain in cytokine recognition (28Dubois S. Magrangeas F. Lehours P. Raher S. Bernard J. Boisteau O. Leroy S. Minvielle S. Godard A. Jacques Y. J. Biol. Chem. 1999; 274: 26978-26984Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Conversely, we show in this study that removal of the C-terminal tail (exons 3-5) of the extracellular part of IL-15Rα (in the context of the sIL-15Rα·IL-2 fusion protein) results in a 10-fold decrease in its binding affinity for IL-15, as seen by SPR, and a 3.5-fold decrease in its affinity as seen in a competition assay. In terms of thermodynamics, the 10-fold decrease in affinity was calculated to correspond to a 10% loss of the free energy of interaction of IL-15 with IL-15Rα. Thus, the N-terminal structural domain encoded by exon 2 (sushi domain) bears most (90%) but not all of the IL-15 binding capacity. Recent data from our laboratory 4E. Mortier, unpublished results. indicate that the domain encoded by exon 3 also contributes to IL-15 binding. The sIL-15Rα-sushi produced in E. coli had an affinity that was 3- to 4-fold lower than that of sIL-15Rα-sushi·IL-2 produced in Chinese hamster ovary cells. This difference cannot be explained by differences in the glycosylation status of the two proteins, inasmuch as the sushi domain does not contain any potential sites for N-or O-linked glycosylations (2Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Crossref PubMed Scopus (970) Google Scholar). It is therefore likely to be