A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells.

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.

IL-15Rα membrane anchorage in either cis or trans is required for stabilization of IL-15 and optimal signaling.

Interleukin (IL)-15 plays an important role in the communication between immune cells. It delivers its signal through different modes involving three receptor chains: IL-15Rα, IL-2Rß and IL-2Rγc. The combination of the different chains result in the formation of IL-15Rα/IL-2Rß/γc trimeric or IL-2Rß/γc dimeric receptors. In this study, we have investigated the role of the IL-15Rα chain in stabilizing the cytokine in the IL-2Rß/γc dimeric receptor. By analyzing the key amino acid residues of IL-15 facing IL-2Rß, we provide evidence of differential interfaces in the presence or in the absence of membrane-anchored IL-15Rα. Moreover, we found that the anchorage of IL-15Rα to the cell surface regardless its mode of presentation - i.e. cis or trans - is crucial for complete signaling. These observations show how the cells can finely modulate the intensity of cytokine signaling through the quality and the level of expression of the receptor chains.

A nanoscale reorganization of the IL-15 receptor is triggered by NKG2D in a ligand-dependent manner.

Natural killer group 2D (NKG2D), an activating receptor on natural killer (NK) cells and a subset of T cells, recognizes stress-inducible proteins, including MICA and ULBP2, which are present on infected or transformed cells. Whether each NKG2D ligand (NKG2DL) has a distinct biological role is not clear. Using superresolution microscopy, we found that NKG2D is constitutively arranged in nanoclusters at the surface of human primary NK cells. Nanoclusters of NKG2D became smaller upon ligation with MICA but became larger upon activation by ULBP2. In addition, ULBP2 induced the reorganization of nanoclusters of the cytokine receptor subunit for both interleukin-2 (IL-2) and IL-15 (IL-2/IL-15Rß), such that these cytokine receptor subunits coalesced with nanoclusters of NKG2D. Functionally, the response of NK cells activated by ULBP2 was augmented by an interaction between ULBP2-bound NKG2D and IL-15R ligated by IL-15 (trans-presented by IL-15Rα-coated surfaces). These data suggest that NKG2DLs are not equivalent in their capacity to activate NKG2D and establish a previously unknown paradigm in how ligand-induced changes to the nanoscale organization of the cell surface can affect immune responses.

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and ß-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.

Interleukin-15 biology and its therapeutic implications in cancer.

Cancer immunotherapy is designed to stimulate the immune system to reject and destroy tumors. Recently, interleukin-15 (IL-15), a member of the four α-helix bundle family of cytokines, has emerged as a candidate immunomodulator for the treatment of cancer. IL-15 acts through its specific receptor, IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes and macrophages. IL-15 exhibits broad activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. It also enhances the cytolytic activity of CD8(+) T cells and induces long-lasting antigen-experienced CD8(+)CD44(hi) memory T cells. IL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces maturation of dendritic cells. It does not stimulate immunosuppressive T regulatory cells (Tregs). Thus, boosting IL-15 activity could enhance innate and specific immunity and fight tumors. Here we review aspects of IL-15 biology that make it a promising agent for anticancer therapy. We also discuss preclinical models in which IL-15 has demonstrated antitumor activity and highlight ongoing clinical trials of IL-15 in patients with cancer and HIV infection.

Interleukin-15:Interleukin-15 receptor α scaffold for creation of multivalent targeted immune molecules.

Human interleukin-15 (hIL-15) and its receptor α (hIL-15Rα) are co-expressed in antigen presenting cells allowing trans-presentation of the cytokine to immune effector cells. We exploited the high-affinity interactions between hIL-15 and the extracellular hIL-15Rα sushi domain (hIL-15RαSu) to create a functional scaffold for the design of multispecific fusion protein complexes. Using single-chain T cell receptors (scTCRs) as recognition domains linked to the IL-15:IL-15Rα scaffold, we generated both bivalent and bispecific complexes. In these fusions, the scTCR domains retain the antigen-binding activity and the hIL-15 domain exhibits receptor binding and biological activity. As expected, bivalent scTCR fusions exhibited improved antigen binding due to increased avidity, whereas fusions comprising two different scTCR domains were capable of binding two cognate peptide/MHC complexes. Bispecific molecules containing scTCR and scCD8αß domains also exhibit enhanced binding to peptide/MHC complexes, demonstrating that the IL-15:IL-15Rα scaffold displays flexibility necessary to support multi-domain interactions with a given target. Surprisingly, functional heterodimeric molecules could be formed by co-expressing the TCR α and ß chains separately as fusions to the hIL-15 and hIL-15RαSu domains. Together, these properties indicate that the hIL-15 and hIL-15RαSu domains can be used as versatile, functional scaffold for generating novel targeted immune molecules.

A biophysical approach to IL-2 and IL-15 receptor function: localization, conformation and interactions.

Interleukin-2 and interleukin-15 (IL-2, IL-15) are key participants in T and NK cell activation and function. Sharing the beta and gamma receptor subunits results in several common functions: e.g. the promotion of T cell proliferation. On the other hand, due to their distinct alpha receptor subunits, they also play opposing roles in immune processes such as activation induced cell death and immunological memory. Divergence of signaling pathways must ensue already at the plasma membrane where the cytokines interact with their receptors. Therefore understanding molecular details of receptor organization and mapping interactions with other membrane proteins that might influence receptor conformation and function, are of key importance. Biophysical/advanced microscopic methods (fluorescence resonance energy transfer (FRET), fluorescence crosscorrelation spectroscopy (FCCS), near-field scanning optical microscopy (NSOM), X-ray crystallography, surface plasmon resonance, NMR spectroscopy) have been instrumental in clarifying the details of receptor structure and organization from the atomic level to the assembly and dynamics of supramolecular clusters. In this short review some important contributions shaping our current view of IL-2 and IL-15 receptors are presented.

Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells.

Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual alpha-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2Ralpha and IL15Ralpha at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2Ralpha and IL15Ralpha at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2Ralpha molecules reside outside the clustered domains, whereas approximately 30% of IL15Ralpha molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2Ralpha and IL15Ralpha domains remained constant, suggesting a 'building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two alpha-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.

Crystal Structure of the interleukin-15.interleukin-15 receptor alpha complex: insights into trans and cis presentation.

Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptor alpha (IL-15Ralpha) from one subset of cells presents IL-15 to neighboring IL-2Rbeta/gammac-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Ralpha. The structure reveals that the alpha receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Ralpha that account for the exceptionally high affinity of the IL-15.IL-15Ralpha complex. Interestingly, analysis of the topology of IL-15 and IL-15Ralpha at the IL-15.IL-15Ralpha interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Ralpha and IL-2Rbeta/gammac expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Ralpha permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15.IL-15Ralpha.IL-2Rbeta.gammac quaternary complex assembly through the use of molecular modeling.