IL-15Rα Complex Research Articles

Abstract 3829: Chronic stimulation with IL-15/IL-15Rα complexes leads to NK cell dysfunction

Abstract IL-15/IL-15Rα complexes induce expansion and activation of cytotoxic lymphocytes including NK and CD8 T cells with higher activity compared to IL-15 alone in vivo, and therefore, have therapeutic potential against cancer and chronic infections. Recent studies demonstrated that continuous treatment with IL-15/IL-15Rα complexes improves the survival of tumor bearing mice, however fail to control tumor outgrowth. Given the limited efficacy of long-term cytokine therapy, we sought to investigate the impact of continuous stimulations with IL-15/IL-15Rα complexes on NK and CD8 T cells in tumor-free mice in vivo. Long-term treatment with IL-15/IL-15Rα complexes induced massive expansion of both NK and CD8 T cells. While the functional capacity of CD8 T cells was enhanced after long-term treatments, NK cells exhibited marked defects in activation, IFNγ production, degranulation, and cytotoxicity. In addition, long-term treatment generated a suppressive environment and resulted in marked increase in Treg and CD11c+Gr1+ cell numbers. However, impaired NK cell function was not due to regulatory T cells or various myeloid cell mediated-suppressive factors. Our results have important clinical implications for the design of immunotherapies and vaccines using multiple doses of immunostimulatory agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3829.

Long-term cytokine therapy with IL-15/IL-15Rα complexes leads to impaired NK cell function (131.11)

Abstract Immunotherapies targeting effector lymphocytes are promising approaches for the treatment of cancer and infectious diseases. One such approach employs IL-15/IL-15Rα complexes which are considerably more potent than IL-15 in expanding cytotoxic lymphocytes including NK and CD8 T cells. Recent studies demonstrated that short-term treatment with IL-15/IL-15Rα complexes is highly effective in driving a transient but substantial reduction in tumor burden via immune-mediated mechanisms whereas continuous cytokine therapy improves survival only modestly and fails to control tumor outgrowth. Given the limited efficacy of long-term therapy, we sought to investigate the impact of continuous in vivo stimulation with IL-15/IL-15Rα complexes on NK and CD8 T cells in tumor-free mice. Long-term treatment with IL-15/IL-15Rα complexes triggered massive expansion of NK and CD8 T cells. Strikingly, however, NK cells exhibited marked defects in activation, IFNγ production, degranulation, and cytotoxicity whereas CD8 T cells showed enhanced functional capacity. Additionally, continuous treatment generated a suppressive environment for transferred naïve NK and CD8 T cells and resulted in accumulation of CD11b+Gr1+ cells. The mechanistic basis for NK cell dysfunction upon chronic stimualation with IL-15/IL-15Ra complexes will be discussed. Our results have important clinical implications for the design of immunotherapies and vaccines using multiple doses of immunostimulatory agents.

Macrophage- and Dendritic-Cell-Derived Interleukin-15 Receptor Alpha Supports Homeostasis of Distinct CD8+ T Cell Subsets

Interleukin-15 receptor alpha (IL-15R alpha) is a pleiotropically expressed molecule that chaperones and trans-presents IL-15 to NK and T cells. To investigate whether IL-15R alpha presented by different cells perform distinct physiological functions, we have generated four lines of mice lacking IL-15R alpha in various cell types. We find that IL-15R alpha expression on macrophages but not dendritic cells (DCs) supports the early transition of antigen specific effector CD8(+) T cells to memory cells. After memory CD8(+) T cell differentiation, IL-15R alpha expression on DCs selectively supports central memory CD8(+) T cells, whereas IL-15R alpha expression on macrophages supports both central and effector memory CD8(+) T cells. By contrast, mice lacking IL-15R alpha on macrophages, DCs, or both, exhibit equivalent defects in NK cell homeostasis and activation. These studies define unique roles for macrophage expression of IL-15R alpha and show that NK cells rely upon distinct IL-15R alpha dependent IL-15 signals than memory CD8(+) T cells. Moreover, they demonstrate the diversity, specification, and geographic restriction of cytokine signals.

IL-15 trans-presentation promotes human NK cell development and differentiation in vivo

The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self–major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2−/−γc−/− mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor α (IL-15Rα) significantly augmented human NK cells. IL-15–IL-15Rα complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16+KIR+ NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56hiCD16−KIR− to CD56loCD16+KIR−, and finally to CD56loCD16+KIR+. These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15–responsive cells during immunotherapy strategies.

How a Cytokine Is Chaperoned through the Secretory Pathway by Complexing with Its Own Receptor: Lessons from Interleukin-15 (IL-15)/IL-15 Receptor α

While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor alpha (IL-15R alpha). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15R alpha and then is secreted. This intracellular IL-15/IL-15R alpha complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15R alpha in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15R alpha offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.

The Exon-3-Encoded Domain of IL-15Rα Contributes to IL-15 High-Affinity Binding and Is Crucial for the IL-15 Antagonistic Effect of Soluble IL-15Rα

We previously showed that a natural soluble form of interleukin-15 (IL-15) Rα corresponding to the full-length ectodomain of IL-15Rα behaved as a potent antagonist of IL-15 action through IL-15Rα/β/γ, whereas a recombinant soluble IL-15Rα sushi domain did not, but instead acted as an agonist of IL-15 action through IL-15Rβ/γ. In order to determine precisely the molecular basis governing these antagonistic versus agonistic actions, we compared the binding properties and biological effects of recombinant soluble IL-15Rα (sIL-15Rα) species containing the sushi domain and different remaining parts of the ectodomain. We first demonstrate that the exon-3-encoded domain and, more particularly, its N-terminal 13-amino-acid (aa) peptide are important, in addition to the adjacent exon-2-encoded sushi domain, for the stabilization of the high-affinity IL-15·IL-15Rα complex by slowing down its dissociation rate and by contributing to about 10–20% of the free energy of interaction. We next show that all sushi-containing sIL-15Rα are agonists on IL-15Rβ/γ, coordinately increasing IL-15 binding and IL-15-induced proliferation. Their agonistic potencies are proportional to their respective affinities for IL-15. We then show that the antagonistic effect of sIL-15Rα in the context of IL-15Rα/β/γ is due to the 13-aa peptide that creates a sterical constraint impeding the binding of the sIL-15Rα·IL-15 complex to the membrane-anchored IL-15Rα/β/γ. In the frame of the soluble IL-15Rα sushi domain–IL-15 fusion protein that contains the 13-aa peptide, this constraint is alleviated as a result of a conformational effect due to the covalent linking of the 13-aa peptide to the N-terminus of IL-15. The soluble IL-15Rα sushi domain–IL-15 fusion protein is therefore able to bind and activate both the IL-15Rβ/γ and the IL-15Rα/β/γ receptors.

IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation

Natural killer (NK) cells are innate immune effectors that mediate rapid responses to viral antigens. Interleukin (IL)-15 and its high affinity IL-15 receptor, IL-15Rα, support NK cell homeostasis in resting animals via a novel trans presentation mechanism. To better understand how IL-15 and IL-15Rα support NK cell activation during immune responses, we have used sensitive assays for detecting native IL-15 and IL-15Rα proteins and developed an assay for detecting complexes of these proteins. We find that IL-15 and IL-15Rα are preassembled in complexes within the endoplasmic reticulum/Golgi of stimulated dendritic cells (DCs) before being released from cells. IL-15Rα is required for IL-15 production by DCs, and IL-15 that emerges onto the cell surface of matured DCs does not bind to neighboring cells expressing IL-15Rα. We also find that soluble IL-15–IL-15Rα complexes are induced during inflammation, but membrane-bound IL-15–IL-15Rα complexes, rather than soluble complexes, support NK cell activation in vitro and in vivo. Finally, we provide in vivo evidence that expression of IL-15Rα specifically on DCs is critical for trans presenting IL-15 and activating NK cells. These studies define an unprecedented cytokine–receptor biosynthetic pathway in which IL-15Rα serves as a chaperone for IL-15, after which membrane-bound IL-15Rα–IL-15 complexes activate NK cells via direct cell–cell contact.

CD8 T cell response to IL‐15/IL‐15Rα complex is MHC class I dependent

Recent findings illustrate that IL‐15 operates via a unique mechanism termed transpresentation. In this system, IL‐15 produced by one cell type is bound to IL‐15Rα expressed by the same cell and presented to apposing cells expressing the IL‐15Rβ/γC complex. We have recently shown that administering soluble IL‐15Rα complexed with IL‐15 can greatly enhance IL‐15 activity by increasing IL‐15 half‐life and forcing transpresentation of IL‐15. Furthermore, IL‐15/IL‐15Rα complex can cause the proliferation and maturation of naïve CD8 T cells. We now show that the CD8 T cell response to exogenous IL‐15/IL‐15Rα complex is MHC class I dependent. In the absence of MHC class I, naïve CD8 T cells scarcely proliferate in response to IL‐15/IL‐15Rα complex. In addition, providing specific peptide along with IL‐15/IL‐15Rα complex can enhance the proliferation of antigen‐specific CD8 T cells. The responding antigen‐specific CD8 T cells undergo an expansion and contraction phase, resulting in a larger frequency of remaining memory‐phenotype cells. These data suggest that IL‐15/IL‐15Rα complex has selective effects on antigen activated CD8 T cells. Our findings have important implications for directing IL‐15/IL‐15Rα complex based therapy to specific antigen targets, and illustrate the possible adjuvant uses of IL‐15/IL‐15Rα complex.NIH Grant AI‐051583 (to L.L.) and NCC Predoctoral Fellowship (to T.A.S.).

Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.

Interleukin 2: from immunostimulation to immunoregulation and back again

Interleukin 2 (IL-2) was one of the first cytokines to be discovered. However, the complex role of IL-2 and its receptor in the regulation of immune responses is only now emerging. This review explores the various signals triggered by IL-2 and discusses their translation into biological function. A model is outlined that accommodates the seemingly contradictory functions of IL-2, and explains how one cytokine can be an essential T-cell growth and differentiation factor and yet also be indispensable to maintain peripheral tolerance.

Crystal structure of the IL-15–IL-15Rα complex, a cytokine-receptor unit presented in trans

Interleukin 15 (IL-15) and IL-2, which promote the survival of memory CD8(+) T cells and regulatory T cells, respectively, bind receptor complexes that share beta- and gamma-signaling subunits. Receptor specificity is provided by unique, nonsignaling alpha-subunits. Whereas IL-2 receptor-alpha (IL-2Ralpha) is expressed together in cis with the beta- and gamma-subunits on T cells and B cells, IL-15Ralpha is expressed in trans on antigen-presenting cells. Here we present a 1.85-A crystal structure of the human IL-15-IL-15Ralpha complex. The structure provides insight into the molecular basis of the specificity of cytokine recognition and emphasizes the importance of water in generating this very high-affinity complex. Despite very low IL-15-IL-2 sequence homology and distinct receptor architecture, the topologies of the IL-15-IL-15Ralpha and IL-2-IL-2Ralpha complexes are very similar. Our data raise the possibility that IL-2, like IL-15, might be capable of being presented in trans in the context of its unique receptor alpha-chain.

Soluble Interleukin (IL)-15Rα Is Generated by Alternative Splicing or Proteolytic Cleavage and Forms Functional Complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

Augmenting the biological activity of IL-15 with soluble IL-15Ralpha (95.21)

Abstract IL-15 is thought to be normally presented in vivo as a cell-associated cytokine bound to IL-15Rα. We show here that the biological activity of soluble IL-15 is much improved after interaction with recombinant soluble IL-15Rα; after injection, soluble IL-15/IL-15Rα complexes rapidly induce strong and selective expansion of memory CD8+ cells and natural killer cells. Surprisingly, we also find that sIL-15/IL-15Rα complexes can drive conversion of naive T cells to memory T cells in vivo, without administration of antigen. This conversion is associated with an acquisition of effector function. The enhancing effect of IL-15Rα binding may explain why IL-15 normally functions as a cell-associated cytokine. Our results suggest the biological activity of IL-15 as a therapeutic reagent could be considerably enhanced by administration of preformed soluble IL-15/IL-15Rα complexes.

The IL-15/IL-15Rα on cell surfaces enables sustained IL-15 activity and contributes to the long survival of CD8 memory T cells

We previously described unique features of the IL-15 receptor (IL-15R)alpha. IL-15Ralpha by itself forms stable complexes with IL-15 on cell surfaces and presents IL-15 in trans to neighboring natural killer/T cells. Moreover, the membrane IL-15/IL-15Ralpha complexes (membIL-15) undergo endosomal internalization but survive lysosomal degradation, allowing the complexes to recycle back to the cell surface. Here, we show that membIL-15+ cells act as a persistent source of IL-15 for the surrounding microenvironment (intercellular reservoir effect). Additionally, membIL-15+ cells give rise to augmented retention of IL-15 in the circulation as well as in tissues. Curiously, IL-15 retention was particularly associated with lungs, rather than with lymph nodes, in normal unstimulated mice, which correlated with the preferential homing of antigen-specific CD8 T cells to lungs during their contraction phase in an IL-15Ralpha-dependent manner. Furthermore, membIL-15, unlike soluble IL-15, caused sustained IL-15 signal transduction in the target cells. Collectively, these characteristics define IL-15 as a unique cytokine with prolonged in vivo survival and sustained biological action on the target cells, which may account for the proposed persistent action of IL-15 that helps the long-term survival of functional CD8 memory T cells in vivo.

Converting IL-15 to a superagonist by binding to soluble IL-15Rα

IL-15 is normally presented in vivo as a cell-associated cytokine bound to IL-15Ralpha. We show here that the biological activity of soluble IL-15 is much improved after interaction with recombinant soluble IL-15Ralpha; after injection, soluble IL-15/IL-15Ralpha complexes rapidly induce strong and selective expansion of memory-phenotype CD8(+) cells and natural killer cells. These findings imply that binding of IL-15Ralpha to IL-15 may create a conformational change that potentiates IL-15 recognition by the betagamma(c) receptor on T cells. The enhancing effect of IL-15Ralpha binding may explain why IL-15 normally functions as a cell-associated cytokine. Significantly, the results with IL-2, a soluble cytokine, are quite different; thus, IL-2 function is markedly inhibited by binding to soluble IL-2Ralpha.

The Structure of the Interleukin-15α Receptor and Its Implications for Ligand Binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.

Membrane-bound and soluble IL-15/IL-15Rα complexes display differential signaling and functions on human hematopoietic progenitors

AbstractMembrane-bound and soluble interleukin-15 (IL-15)/IL-15 receptor α (Rα) complexes trigger differential transcription factor activation and functions on human hematopoietic progenitors. Indeed, human spleen myofibroblasts (SMFs) are characterized by a novel mechanism of IL-15 trans-presentation (SMFmb [membrane-bound]-IL-15), based on the association of an endogenous IL-15/IL-15Rα complex with the IL-15Rβγc chains. SMFmb-IL-15 (1) induces lineage-specific signaling pathways that differ from those controlled by soluble IL-15 in unprimed and committed normal progenitors; (2) triggers survival and proliferation of leukemic progenitors expressing low-affinity IL-15R (M07Sb cells); (3) causes only an antiapoptotic effect on leukemic cells expressing high-affinity receptors (TF1β cells). This behavior is likely due to the IL-15Rα chain present on these cells that interact with the SMFmb-IL-15, inhibiting signal transducer and transcriptional activator 5 (STAT5) activation. On the other hand, the soluble IL-15/IL-15Rα complex (hyper IL-15) displays a dominant pattern of action, activating only those cells expressing low-affinity IL-15R (IL-15Rβγc). Thus, hyper IL-15 induces antiapoptotic effects on M075b cells and the up-regulation of STAT6 activation on adult peripheral blood (PB) pre-natural killer (NK) committed progenitors. The latter effect using 100-fold concentrations of recombinant (r)-IL-15. In conclusion, SMFmb-IL-15 and soluble IL-15Rα/IL-15 complexes seem to play a pivotal role in the control of the survival, proliferation and differentiation of both normal and leukemic circulating progenitors, highlighting new functions of IL-15 and of IL-15Rα.

IL-15Rα Recycles and Presents IL-15 In trans to Neighboring Cells

We report intriguing aspects of the contribution of IL-15Rα to IL-15 functions. Consistent with high-affinity interactions between IL-15 and IL-15Rα, these two molecules form stable complexes on the cell surface of activated monocytes. The formation of IL-15/IL-15Rα complexes on cell surfaces induces a trans-endosomal recycling of IL-15 leading to the persistence of surface-bound IL-15 due to the constantreappearance of IL-15 on plasma membranes. This complex contributes to the long survival of T cells expressing IL-15Rα after IL-15 withdrawal. Finally, these complexes on activated monocytes present IL-15 in trans to target cells such as CD8 + T cells that express only IL-2/15Rβ and γc upon cell-cell interaction.

Natural Splicing of Exon 2 of Human Interleukin-15 Receptor α-Chain mRNA Results in a Shortened Form with a Distinct Pattern of Expression

We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15Ralpha) transcripts resulting from exon-splicing mechanisms within the IL-15Ralpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Ralpha bound IL-15 with high affinity, Delta2IL-15Ralpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ralpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15.IL-15Ralpha complex at the nuclear level. In sharp contrast, Delta2IL-15Ralpha was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Ralpha is a natural process that might play regulatory roles at different levels.