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.

Skeletal muscle IL-15/IL-15Rα and myofibrillar protein synthesis after resistance exercise.

In vitro and in vivo studies described the myokine IL-15 and its receptor IL-15Rα as anabolic/anti-atrophy agents, however, the protein expression of IL-15Rα has not been measured in human skeletal muscle and data regarding IL-15 expression remain inconclusive. The purpose of the study was to determine serum and skeletal muscle IL-15 and IL-15Rα responses to resistance exercise session and to analyze their association with myofibrillar protein synthesis (MPS). Fourteen participants performed a bilateral leg resistance exercise composed of four sets of leg press and four sets of knee extension at 75% 1RM to task failure. Muscle biopsies were obtained at rest, 0, 4 and 24 hours post-exercise and blood samples at rest, mid-exercise, 0, 0.3, 1, 2, 4 and 24 hours post-exercise. Serum IL-15 was increased by ~5.3-fold immediately post-exercise, while serum IL-15Rα decreased ~75% over 1 hour post-exercise (P<.001). Skeletal muscle IL-15Rα mRNA and protein expression were increased at 4 hours post-exercise by ~2-fold (P<.001) and ~1.3-fold above rest (P=.020), respectively. At 24 hours post-exercise, IL-15 (P=.003) and IL-15Rα mRNAs increased by ~2-fold (P=.002). Myofibrillar fractional synthetic rate between 0-4 hours was associated with IL-15Rα mRNA at rest (r=.662, P=.019), 4 hours (r=.612, P=.029), and 24 hours post-exercise (r=.627, P=.029). Finally, the muscle IL-15Rα protein up-regulation was related to Leg press 1RM (r=.688, P=.003) and total weight lifted (r=.628, P=.009). In conclusion, IL-15/IL-15Rα signaling pathway is activated in skeletal muscle in response to a session of resistance exercise.

Energy-sensing factors coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase control expression of inflammatory mediators in liver: induction of interleukin 1 receptor antagonist.

Obesity and insulin resistance are associated with chronic, low grade inflammation. Moreover, regulation of energy metabolism and immunity are highly integrated. We hypothesized that energy-sensitive coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK) may modulate inflammatory gene expression in liver. Microarray analysis revealed that PGC-1α up-regulated expression of several cytokines and cytokine receptors, including interleukin 15 receptor α (IL15Rα) and, even more importantly, anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). Overexpression of PGC-1α and induction of PGC-1α by fasting, physical exercise, glucagon, or cAMP was associated with increased IL1Rn mRNA and protein expression in hepatocytes. Knockdown of PGC-1α by siRNA down-regulated cAMP-induced expression of IL1Rn in mouse hepatocytes. Furthermore, knockdown of peroxisome proliferator-activated receptor α (PPARα) attenuated IL1Rn induction by PGC-1α. Overexpression of PGC-1α, at least partially through IL1Rn, suppressed interleukin 1ß-induced expression of acute phase proteins, C-reactive protein, and haptoglobin. Fasting and exercise also induced IL15Rα expression, whereas glucagon and cAMP resulted in reduction in IL15Rα mRNA levels. Finally, AMPK activator metformin and adenoviral overexpression of AMPK up-regulated IL1Rn and down-regulated IL15Rα in primary hepatocytes. We conclude that PGC-1α and AMPK alter inflammatory gene expression in liver and thus integrate energy homeostasis and inflammation. Induction of IL1Rn by PGC-1α and AMPK may be involved in the beneficial effects of exercise and caloric restriction and putative anti-inflammatory effects of metformin.

Cerebral interleukin-15 shows upregulation and beneficial effects in experimental autoimmune encephalomyelitis.

Interleukin (IL)-15 can cross the blood-brain barrier to act on its specific brain receptor (IL15Ralpha) and co-receptors. The important roles of neuronal IL15 and IL15Ralpha in experimental autoimmune encephalomeylitis (EAE) are suggested by the upregulation of IL15Ralpha mRNA in different regions of the brain and spinal cord, and by double-labeling immunohistochemistry showing neuronal localization of IL15 and IL15Ralpha in different neurons. Contrary to expectations, IL15 treatment lessened EAE severity. IL15 knockout mice showed heightened susceptibility to EAE with significantly higher scores that were decreased by treatment with IL15. Thus, IL15 improves this CNS autoimmune disorder as a potential therapeutic agent.

Expression and signaling of novel IL15Ralpha splicing variants in cerebral endothelial cells of the blood-brain barrier.

Interleukin (IL)-15 and its receptors in cerebral microvascular endothelial cells play an important role in mediating neuroinflammatory signaling across the blood-brain barrier. Although alternative splice variants of IL15Ralpha (the specific receptor) are seen in immune cells, the presence and functions of splice variants have not been studied in the cerebral endothelia that compose the blood-brain barrier. In this study, we identified five splice variants from mouse cerebral capillaries by RT-PCR, cloning, and DNA sequencing, and performed domain analysis. Four of these isoforms have never been described in any tissue. All isoforms were detected by qPCR in enriched mouse cerebral microvessels and their expression was increased by tumor necrosis factor treatment in vivo. To determine their functions, plasmids encoding individual isoforms were transfected into RBE4 cerebral endothelial cells. All of these predicted alkalinic proteins were expressed and most showed post-translational modifications. There were variations in their subcellular distribution. Only the full length IL15Ralpha and to a lesser degree isoform alpha1 were trafficked to the cell surface 24 h after over-expression. As shown by a luciferase reporter for signal transducer and activator of transcription (STAT)-3, over-expression of isoforms alpha2 and alpha4 reduced basal STAT3 activation. In comparison with the control, over-expression of the full length IL15Ralpha had a greater effect in increasing IL15-induced STAT3 transactivation than other isoforms. The results show that IL15 signaling in cerebral endothelia is probably an orchestrated effect of all IL15Ralpha splice variants that determine the eventual outcome by differential regulation.

IL-21 preferentially enhances IL-15-mediated homeostatic proliferation of human CD28+ CD8 memory T cells throughout the adult age span.

An age-related decline in human immune response is marked by the accumulation of CD28(-) CD8 T cells, which is attributed to repeated antigenic stimulation and to homeostatic proliferation mediated by cytokines such as IL-15. However, the identity of the cytokines that are responsible for the maintenance of CD28 expression is less known. Here, we report the role of IL-21 in the regulation of IL-15-mediated growth and CD28 expression of CD8 memory T cells of young and old donors. We showed that IL-21 drives more IL-15-stimulated cells to enter cell division and to undergo apoptosis. Furthermore, IL-21 preferentially enhanced IL-15-induced proliferation of CD28(+) CD8 memory T cells over their CD28(-) counterparts, as CD28(+) cells expressed higher levels of IL-15R and IL-21R and greater pSTAT5 upon IL-15 and IL-21 stimulation. In addition, IL-21 reduced IL-15-induced CD28 down-regulation in CD8 memory T cells. Finally, the ability of proliferation and survival in response to homeostatic cytokines IL-15 and IL-21 of CD28(+) CD8 memory T cells was well-maintained with age. Together, these findings suggest that IL-21 enhances IL-15-mediated proliferation of CD8 memory T cells, particularly CD28(+) memory T cells, and also serves as an antagonist to the IL-15-induced increase of CD28(-) CD8 T cells.

High-dose cyclophosphamide-mediated anti-tumor effects by the superior expansion of CD44(high) cells after their selective depletion.

As alkylating agents, cyclophosphamides (CTX) are used to treat various cancers and, ironically, to boost immune responses. In the present study, we attempted to elucidate the mechanism responsible for the immunomodulatory effect of high-dose CTX in an established tumor model. A single injection of high-dose CTX increased the survival rate of immunocompetent, but not immunodeficient, mice. Notably, 10 days after CTX injection, the number of CD44(high) memory T cells significantly increased, without a selective decrease in the actual number and percentage of CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the proportion of Tregs among CD4+ T cells decreased due to expansion of memory and other CD4+ T cell subtypes. This outcome was accompanied by an increase in IL-15 mRNA and up-regulation of IL-15 receptors in the CD44+CD8+ T cell compartment. We postulate that the CTX-induced change in T cell balance may increase anti-tumor immunity.

Elevated expression of transmembrane IL-15 in immune cells correlates with the development of murine lupus: a potential target for immunotherapy against SLE.

Presentation in trans by the Interleukin-15 receptor alpha chain (IL-15Ralpha) has been suggested as the main mechanism for IL-15 anchoring to the cell surface, but it is also evident that IL-15 can exist as a transmembrane protein. We herein demonstrate that replacement of the first 41 residues of human IL-15 (hIL-15) with Igkappa chain leader sequence resulted in secretion of most of the recombinant hIL-15 expressed in transfectant cells, thus identifying the transmembrane region of IL-15. A fusion protein (hIL-15Ralpha-Fc) between the extracellular domain of hIL-15Ralpha and the Fc fragment of IgG1 was prepared and shown to be able to bind with transmembrane IL-15 (tmIL-15). The level of tmIL-15 expression in macrophages, activated T cells and B cells from 6-month-old BXSB male mice, an animal model for systemic lupus erythematosus (SLE), was significantly increased compared with that from BXSB females or young males. In addition, hIL-15Ralpha-Fc was able to block the T cell stimulating and anti-apoptotic effect of the tmIL-15-positive BXSB macrophages in vitro. Intravenous administration of hIL-15Ralpha-Fc reduced the titre of autoantibodies against dsDNA and also proteinuria in aged BXSB males, implying that neutralization of IL-15 activity in vivo may be an effective way of treating SLE.

Cell-specific interleukin-15 and interleukin-15 receptor subunit expression and regulation in pneumococcal pneumonia--comparison to chlamydial lung infection.

Interleukin (IL)-15 has critical impact on the homeostasis and activation of natural killer cells, natural killer T cells, gammadeltaT cells, and CD8(+)T cells, and contributes to antimicrobial defenses particularly at mucosal sites. The respiratory tract comprises a large mucosal surface and harbors significant amounts of lymphocytes, however the expression pattern of IL-15 in the lung and its role in local immune responses are largely unknown. We therefore analyzed the differential expression of IL-15 and the IL-15 receptor (IL-15R) complex in the lungs of mice and demonstrated substantial constitutive expression in bronchial and alveolar epithelial cells, alveolar macrophages, and vascular smooth muscle cells, implicating contribution to pulmonary immune cell homeostasis already under normal conditions. The induction of pneumococcal pneumonia but not the infection with Chlamydophila pneumoniae evoked a significant up-regulation of IL-15 on alveolar macrophages and bronchial epithelial cells, with the latter presenting de-novo expression of IL-15 on their basolateral surface and additional up-regulation of IL-15Ralpha. Moreover, transcriptome analysis as well as semi-quantitative PCR indicated at least partial transcriptional regulation in mice lungs. In conclusion IL-15 is suggested being of functional importance in the pulmonary immune response against pneumococcal pneumonia.

Soluble Interleukin IL-15Ralpha 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.

Juxtacrine function of interleukin-15/interleukin-15 receptor system in tumour derived human B-cell lines.

Interleukin-15 (IL-15) is a cytokine that induces proliferation and promotes cell survival of human T, B and NK cells. IL-15 and interleukin-2 (IL-2) exhibit a similar spectrum of immune effects and share the IL-2 receptor (IL-2R) subunits IL-2Rbeta and IL-2Rgamma(c) for signalling in haematopoietic cells. Furthermore, each cytokine has a private alpha receptor, namely IL-2Ralpha for IL-2 and IL-15Ralpha for IL-15, that functions in ligand binding. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods, the expression and secretion of IL-15 and IL-15Ralpha in tumour-derived B-cell lines were studied. The results as presented in this study identify that IL-15 mRNA is predominantly expressed in EBV positive (EBV(+)) B-cell lines, although IL-15Ralpha is ubiquitously and constitutively expressed in all these B-cell lines. Although no detectable levels of IL-15 protein secretion were observed in any of these cell lines, we were able to detect membrane-bound expression of IL-15 protein by FACS analysis in some cell lines. These data imply that the IL-15/IL-15R system requires complex regulatory mechanisms for protein secretion. Taken together, we speculate that these results suggest a juxtacrine, intracrine function for IL-15/IL-15R.

Growth and apoptosis of human natural killer cell neoplasms: role of interleukin-2/15 signaling.

Interleukin (IL)-15 plays an important role in the survival of human natural killer (NK) cells. We investigated IL-2/15 signaling in NK cell neoplasms from five patients and in five cell lines (NK-92, KHYG-1, SNK-6, HANK1 and MOTN-1) compared to mature peripheral NK cells from 10 healthy subjects. Apoptosis of NK cell lines was prevented by addition of IL-15 in vitro. Blocking IL-2/15Rbeta on IL-2-stimulated NK-92 cells resulted in reduced expression of Bcl-X(L) and phosphorylated Stat5, which paralleled early apoptosis without altering Bcl-2 expression. These data add IL-2/15Rbeta to the list of factors important for the survival of NK cell neoplasms.