Selective Targeting of IL-15Rα Is Sufficient to Reduce Inflammation.

Cytokines are crucial molecules for maintaining the proper functioning of the immune system. Nevertheless, a dysregulation of cytokine expression could be involved in the pathogenesis of autoimmune diseases. Interleukin (IL)-15 is a key factor for natural killer cells (NK) and CD8 T cells homeostasis, necessary to fight cancer and infections but could also be considered as a pro-inflammatory cytokine involved in autoimmune inflammatory disease, including rheumatoid arthritis, psoriasis, along with tumor necrosis factor alpha (TNF-α), IL-6, and IL-1ß. The molecular mechanisms by which IL-15 exerts its inflammatory function in these diseases are still unclear. In this study, we generated an IL-15-derived molecule called NANTIL-15 (New ANTagonist of IL-15), designed to selectively inhibit the action of IL-15 through the high-affinity trimeric IL-15Rα/IL-2Rß/γc receptor while leaving IL-15 signaling through the dimeric IL-2Rß/γc receptor unaffected. Administrating of NANTIL-15 in healthy mice did not affect the IL-15-dependent cell populations such as NK and CD8 T cells. In contrast, we found that NANTIL-15 efficiently reduced signs of inflammation in a collagen-induced arthritis model. These observations demonstrate that the inflammatory properties of IL-15 are linked to its action through the trimeric IL-15Rα/IL-2Rß/γc receptor, highlighting the interest of selectively targeting this receptor.

IL-15 superagonist RLI has potent immunostimulatory properties on NK cells: implications for antimetastatic treatment.

BACKGROUND: As the immune system is compromised in patients with cancer, therapeutic strategies to stimulate immunity appear promising, to avoid relapse and increase long-term overall survival. Interleukin-15 (IL-15) has similar properties to IL-2, but does not cause activation-induced cell death nor activation and proliferation of regulatory T cells (Treg), which makes it a serious candidate for anticancer immunotherapy. However, IL-15 has a short half-life and high doses are needed to achieve responses. Designed to enhance its activity, receptor-linker-IL-15 (RLI) (SO-C101) is a fusion molecule of human IL-15 covalently linked to the human IL-15Rα sushi+ domain currently assessed in a phase I/Ib clinical trial on patients with advanced/metastatic solid cancer. METHODS: We investigated the antimetastatic activity of RLI in a 4T1 mouse mammary carcinoma that spontaneously metastasizes and evaluated its immunomodulatory role in the metastatic lung microenvironment. We further characterized the proliferation, maturation and cytotoxic functions of natural killer (NK) cells in tumor-free mice treated with RLI. Finally, we explored the effect of RLI on human NK cells from healthy donors and patients with non-small cell lung cancer (NSCLC). RESULTS: RLI treatment displayed antimetastatic properties in the 4T1 mouse model. By characterizing the lung microenvironment, we observed that RLI restored the balance between NK cells and neutrophils (CD11b+ Ly6Ghigh Ly6Clow) that massively infiltrate lungs of 4T1-tumor bearing mice. In addition, the ratio between NK cells and Treg was strongly increased by RLI treatment. Further pharmacodynamic studies in tumor-free mice revealed superior proliferative and cytotoxic functions on NK cells after RLI treatment compared with IL-15 alone. Characterization of the maturation stage of NK cells demonstrated that RLI favored accumulation of CD11b+ CD27high KLRG1+ mature NK cells. Finally, RLI demonstrated potent immunostimulatory properties on human NK cells by inducing proliferation and activation of NK cells from healthy donors and enhancing cytotoxic responses to NKp30 crosslinking in NK cells from patients with NSCLC. CONCLUSIONS: Collectively, our work demonstrates superior activity of RLI compared with rhIL-15 in modulating and activating NK cells and provides additional evidences for a therapeutic strategy using RLI as antimetastatic molecule.

Emergence of NK Cell Hyporesponsiveness after Two IL-15 Stimulation Cycles.

IL-15 is a cytokine playing a crucial role in the function of immune cells, including NK and CD8 T cells. In this study, we demonstrated that in vivo, in mice, IL-15-prestimulated NK cells were no longer able to respond to a second cycle of IL-15 stimulation. This was illustrated by defects in cell maturation, proliferation, and activation, seemingly linked to the environment surrounding NK cells but not related to the presence of CD4 regulatory T cells, TGF-ß, or IL-10. Moreover, NK cells from immunodeficient mice could respond to two cycles of IL-15 stimulation, whereas an adoptive transfer of CD44+CD8+ cells impaired their responsiveness to the second cycle. Conversely, in immunocompetent mice, NK cell responsiveness to a second IL-15 stimulation was restored by the depletion of CD8+ cells. These biological findings refine our understanding of the complex mode of action of NK cells in vivo, and they should be taken into consideration for IL-15-based therapy.

[Interleukin 2 revival: a revisited model and new therapeutic applications]. / Le renouveau de l'interleukine 2 - Modèle revisité et nouvelles applications thérapeutiques.

Interleukin-2, a cytokine identified as T-cell growth factor, has long been regarded as central to the development and effector activities of immune responses. Several gene knockout mouse studies and observations in humans, however, have undermined that vision, and the discovery of regulatory T cells showed that IL-2, in contrast to the accepted dogma, has the essential function of promoting (1) homeostasis and (2) the function of these T regulator cells the which, limit the action of the effector cells, in particular to prevent the autoimmune reaction drifts. This new paradigm has major implications on the use of IL-2 in therapy, and creates new strategies to manipulate the Teffectors/Tregulators balance.

IL-15 Trans-Signaling with the Superagonist RLI Promotes Effector/Memory CD8+ T Cell Responses and Enhances Antitumor Activity of PD-1 Antagonists.

Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity.

Syndecan-1 regulates the biological activities of interleukin-34.

IL-34 is a challenging cytokine sharing functional similarities with M-CSF through M-CSFR activation. It also plays a singular role that has recently been explained in the brain, through a binding to the receptor protein tyrosine phosphatase RPTPß/ζ. The aim of this paper was to look for alternative binding of IL-34 on other cell types. Myeloid cells (HL-60, U-937, THP-1) were used as cells intrinsically expressing M-CSFR, and M-CSFR was expressed in TF-1 and HEK293 cells. IL-34 binding was studied by Scatchard and binding inhibition assays, using 125I-radiolabelled cytokines, and surface plasmon resonance. M-CSFR activation was analysed by Western blot after glycosaminoglycans abrasion, syndecan-1 overexpression or repression and addition of a blocking anti-syndecan antibody. M-CSF and IL-34 induced different patterns of M-CSFR phosphorylations, suggesting the existence of alternative binding for IL-34. Binding experiments and chondroitinase treatment confirmed low affinity binding to chondroitin sulphate chains on cells lacking both M-CSFR and RPTPß/ζ. Amongst the proteoglycans with chondroitin sulphate chains, syndecan-1 was able to modulate the IL-34-induced M-CSFR signalling pathways. Interestingly, IL-34 induced the migration of syndecan-1 expressing cells. Indeed, IL-34 significantly increased the migration of THP-1 and M2a macrophages that was inhibited by addition of a blocking anti-syndecan-1 antibody. This paper provides evidence of alternative binding of IL-34 to chondroitin sulphates and syndecan-1 at the cell surface that modulates M-CSFR activation. In addition, IL-34-induced myeloid cell migration is a syndecan-1 dependent mechanism.

Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse.

Rituximab (RTX), a chimeric IgG1 monoclonal antibody directed against the CD20 antigen, has revolutionized the treatment of B-cell malignancies. Nevertheless, the relapsed/refractory rates are still high. One strategy to increase the clinical effectiveness of RTX is based on antibody-cytokine fusion protein (immunocytokine; ICK) vectorizing together at the tumor site the antibody effector activities and the cytokine co-signal required for the generation of cytotoxic cellular immunity. Such ICKs linking various antibody formats to interleukin (IL)-2 are currently being investigated in clinical trials and have shown promising results in cancer therapies. IL-15, a structurally-related cytokine, is now considered as having a better potential than IL-2 in antitumor immunotherapeutic strategies. We have previously engineered the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. Compared with IL-15, RLI displayed better biological activities in vitro and higher antitumor effects in vivo in murine and human cancer models. In this study, we investigated the advantages of fusing RLI to RTX. Anti-CD20-RLI kept its binding capacity to CD20, CD16 and IL-15 receptor and therefore fully retained both antibody effector functions (ADCC and CDC), and the cytokine potential of RLI. In a severe combined immunodeficiency (SCID) mouse model of disseminated residual lymphoma, anti-CD20-RLI was found to induce long-term survival of 90% of mice up to at least 120 days whereas RLI and RTX, alone or in combination, just delayed the disease onset (100% of death at 28, 40 and 51 days respectively). These findings suggest that such ICK could improve the clinical efficacy of RTX, particularly in patients with refractory B-cell lymphoma.

IL-15.IL-15Rα complex shedding following trans-presentation is essential for the survival of IL-15 responding NK and T cells.

Interleukin (IL)-15 and its specific receptor chain, IL-15Rα, support the development of various effector cells, including NK and CD8 T cells via a mechanism called trans-presentation. Whereas the dynamic of trans-presentation has been shown to involve the recycling of IL-15Rα by presenting cells, the way responding cells integrate, or take advantage of this process has not been evaluated yet. To address this question, we set up a trans-presentation model using a membrane-bound IL-15.IL-15Rα fusion protein, and found that IL-15 is detectable within responding cells following IL-15 trans-presentation. The role of the proteolytic cleavage of IL-15Rα in this process was investigated by generating an uncleavable form of IL-15Rα. We showed that IL-15 entry into responding cells necessitates the cleavage of IL-15.IL-15Rα complex from the surface of IL-15 presenting cells, and observed that IL-15Rα cleavage is associated with a decrease of the duration of Stat5 signaling. Once separated from presenting cells, responding cells are able to recycle IL-15.IL-15Rα complexes via intracellular compartments, for residual proliferation in a time-limited manner. These studies define an unprecedented cytokine pathway in which the IL-15.IL-15Rα complex cleaved from presenting cells allows responding cells to internalize, store and use IL-15.IL-15Rα complex for their own proliferation and survival.

Regulation of CD4(+)NKG2D(+) Th1 cells in patients with metastatic melanoma treated with sorafenib: role of IL-15Rα and NKG2D triggering.

Beyond cancer-cell intrinsic factors, the immune status of the host has a prognostic impact on patients with cancer and influences the effects of conventional chemotherapies. Metastatic melanoma is intrinsically immunogenic, thereby facilitating the search for immune biomarkers of clinical responses to cytotoxic agents. Here, we show that a multi-tyrosine kinase inhibitor, sorafenib, upregulates interleukin (IL)-15Rα in vitro and in vivo in patients with melanoma, and in conjunction with natural killer (NK) group 2D (NKG2D) ligands, contributes to the Th1 polarization and accumulation of peripheral CD4(+)NKG2D(+) T cells. Hence, the increase of blood CD4(+)NKG2D(+) T cells after two cycles of sorafenib (combined with temozolomide) was associated with prolonged survival in a prospective phase I/II trial enrolling 63 patients with metastatic melanoma who did not receive vemurafenib nor immune checkpoint-blocking antibodies. In contrast, in metastatic melanoma patients treated with classical treatment modalities, this CD4(+)NKG2D(+) subset failed to correlate with prognosis. These findings indicate that sorafenib may be used as an "adjuvant" molecule capable of inducing or restoring IL-15Rα/IL-15 in tumors expressing MHC class I-related chain A/B (MICA/B) and on circulating monocytes of responding patients, hereby contributing to the bioactivity of NKG2D(+) Th1 cells.

Antitumor activity of an immunocytokine composed of an anti-GD2 antibody and the IL-15 superagonist RLI.

The use of interleukin (IL)-15 or the IL-15 superagonist RLI as immunological adjuvants presents many advantages over that of IL-2, including a reduced toxicity and an improved efficacy. We have generated an immunocytokine that specifically targets RLI to a tumor-associated antigen, namely, disialoganglioside GD2. This agent displayed robust antitumor activity in 2 syngeneic murine models of malignant disease.

Tumor targeting of the IL-15 superagonist RLI by an anti-GD2 antibody strongly enhances its antitumor potency.

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor-associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)-2-based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL-2 in vitro, IL-15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL-2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. RLI showed better biological activities than IL-15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti-GD2-RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti-GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

Autonomous and extrinsic regulation of thymopoiesis in human immune system (HIS) mice.

Human Immune System (HIS) mice represent a novel biotechnology platform to dissect human haematopoiesis and immune responses. However, the limited human T-cell development that is observed in HIS mice restricts its utility for these applications. Here, we address whether reduced thymopoiesis in HIS mice reflects an autonomous defect in T-cell precursors and/or a defect in the murine thymic niche. Human thymocyte precursors seed the mouse thymus and their reciprocal interactions with murine thymic epithelial cells (TECs) led to both T-cell and TEC maturation. The human thymocyte subsets observed in HIS mice demonstrated survival, proliferative and phenotypic characteristics of their normal human counterparts, suggesting that the intrinsic developmental program of human thymocytes unfolds normally in this xenograft setting. We observed that exogenous administration of human IL-15/IL-15Rα agonistic complexes induced the survival, proliferation and absolute numbers of immature human thymocyte subsets, without any obvious effect on cell-surface phenotype or TCR Vß usage amongst the newly selected mature single-positive (SP) thymocytes. Finally, when IL-15 was administered early after stem cell transplantation, we noted accelerated thymopoiesis resulting in the more rapid appearance of peripheral naïve T cells. Our results highlight the functional capacity of murine thymic stroma cells in promoting human thymopoiesis in HIS mice but suggest that the "cross-talk" between murine thymic stroma and human haematopoietic precursors may be suboptimal. As IL-15 immunotherapy promotes early thymopoiesis, this novel approach could be used to reduce the period of T-cell immunodeficiency in the post-transplant clinical setting.

Antibody-Hapten Recognition at the Surface of Functionalized Liposomes Studied by SPR: Steric Hindrance of Pegylated Phospholipids in Stealth Liposomes Prepared for Targeted Radionuclide Delivery.

Targeted PEGylated liposomes could increase the amount of drugs or radionuclides delivered to tumor cells. They show favorable stability and pharmacokinetics, but steric hindrance of the PEG chains can block the binding of the targeting moiety. Here, specific interactions between an antihapten antibody (clone 734, specific for the DTPA-indium complex) and DTPA-indium-tagged liposomes were characterized by surface plasmon resonance (SPR). Non-PEGylated liposomes fused on CM5 chips whereas PEGylated liposomes did not. By contrast, both PEGylated and non-PEGylated liposomes attached to L1 chips without fusion. SPR binding kinetics showed that, in the absence of PEG, the antibody binds the hapten at the surface of lipid bilayers with the affinity of the soluble hapten. The incorporation of PEGylated lipids hinders antibody binding to extents depending on PEGylated lipid fraction and PEG molecular weight. SPR on immobilized liposomes thus appears as a useful technique to optimize formulations of liposomes for targeted therapy.

IL-15 transpresentation promotes both human T-cell reconstitution and T-cell-dependent antibody responses in vivo.

Cytokine immunotherapies targeting T lymphocytes are attractive clinical interventions against viruses and tumors. In the mouse, the homeostasis of memory α/ß CD8(+) T cells and natural killer (NK) cells is significantly improved with increased IL-15 bioavailability. In contrast, the role of "transpresented" IL-15 on human T-cell development and homeostasis in vivo is unknown. We found that both CD8 and CD4 T cells in human immune system (HIS) mice are highly sensitive to transpresented IL-15 in vivo, with both naïve (CD62L(+)CD45RA(+)) and memory phenotype (CD62L(-)CD45RO(+)) subsets being significantly increased following IL-15 "boosting." The unexpected global improvement in human T-cell homeostasis involved enhanced proliferation and survival of both naïve and memory phenotype peripheral T cells, which potentiated B-cell responses by increasing the frequency of antigen-specific responses following immunization. Transpresented IL-15 did not modify T-cell activation patterns or alter the global T-cell receptor (TCR) repertoire diversity. Our results indicate an unexpected effect of IL-15 on human T cells in vivo, in particular on CD4(+) T cells. As IL-15 promotes human peripheral T-cell homeostasis and increases the frequency of neutralizing antibody responses in HIS mice, IL-15 immunotherapy could be envisaged as a unique approach to improve vaccine responses in the clinical setting.

Different dynamics of IL-15R activation following IL-15 cis- or trans-presentation.

Interleukin (IL)-15 is a cytokine critical for the homeostasis and the function of NK cells, NK-T cells, and memory CD8+ T cells. IL-15 signals are delivered through the IL-15Rß and the common γ (γ(c)) receptor chains. The third receptor chain, IL-15Rα, confers specificity and high affinity for the cytokine. While IL-15 can activate with high affinity the trimeric receptor expressed by a target cell (cis-presentation), IL-15Rα is also known to trans-present IL-15 with high affinity to target cells expressing the IL-15Rß/γ(c) complex. In order to compare the IL-15 cis- and trans-presentation processes, and using a T cell line expressing both IL-15Rα/ß/γ(c) and IL-15Rß/γ(c), we analyzed cell surface receptor chain down-modulation, cytokine internalization and signaling responses induced either with IL-15 (cis-presentation) or with RLI, a protein resulting from fusion between IL-15 and an extended IL-15Rα sushi domain, that mimics trans-presentation. Whereas IL-15 bound with high affinity to IL-15Rα/ß/γ(c), RLI bound with a similar high affinity to IL-15Rß/γ(c). The kinetics of cell surface IL-15R down-modulation were slower following RLI treatment than after IL-15 treatment, as were the kinetics of RLI internalization, which was slower than that of IL-15. IL-15 and RLI dose-dependently induced the activation of similar signaling pathways. However, the kinetics and duration of these activations were markedly different, RLI-induced signaling, being slower, but more prolonged than that induced by IL-15, although the final proliferative responses at 48 h were similar. These findings collectively indicate that IL-15 cis- and trans-presentation mechanisms lead to different dynamics of receptor activation and signal transduction, with cis-presentation inducing fast and transient responses, and trans-presentation inducing slower, more persistent ones. They provide clues for a better understanding of how IL-15 action is controlled, and how it plays a key role in the coordination between innate and adaptative immunity.

Interleukin-15 and its soluble receptor mediate the response to infliximab in patients with Crohn's disease.

BACKGROUND & AIMS: Infliximab is a monoclonal antibody against tumor necrosis factor that is used to treat patients with inflammatory bowel disease. We investigated serum levels and cellular expression of interleukin (IL)-15 and its receptor (sIL-15Ralpha) in patients with Crohn's disease (CD) treated with infliximab; and the effect on sIL-15Ralpha secretion by epithelial cells. METHODS: CD patients were given infliximab (n = 40; 3 infusions); 37 healthy controls were studied. Serum levels of IL-15, sIL-15Ralpha, and complex were determined by radioimmunoassay and cytokine levels by enzyme-linked immunosorbent assay. IL-15Ralpha and A Desintegrin and Metalloproteinase 17 levels were assessed by immunohistochemistry. Epithelial cell lines (HT-29 and Caco-2) were cultured with infliximab, adalimumab, or etanercept. Patients were classified as responders and nonresponders according to their Crohn's Disease Activity Index and clinical observations. RESULTS: Before infliximab, IL-15 was higher in responders than in controls and nonresponders. After infliximab, IL-15 decreased in responders while remaining stable in nonresponders. sIL-15Ralpha and IL-15/sIL-15Ralpha complex levels were higher in CD than in controls and increased only in responders after infliximab. IL-15Ralpha and A Desintegrin and Metalloproteinase 17 colocalized in epithelial cells and were higher in CD patients. In vitro, infliximab but not adalimumab and etanercept induced sIL-15Ralpha secretion by epithelial cells. CONCLUSIONS: Serum level of sIL-15Ralpha and the IL-15/sIL-15Ralpha complex increased in responder patients and the response was associated with a decrease of IL-15. Infliximab induced the release of the IL-15 receptor alpha, suggesting a specific modulation of IL-15 and its soluble receptor by reverse signaling through transmembrane tumor necrosis factor alpha.

High antitumor activity of RLI, an interleukin-15 (IL-15)-IL-15 receptor alpha fusion protein, in metastatic melanoma and colorectal cancer.

Interleukin (IL)-15 has an important role in tumor immunosurveillance and has a contemplated use in tumor immunotherapy. We have previously engineered the fusion protein RLI, composed of the NH(2)-terminal (amino acids 1-77, sushi+) domain of IL-15 receptor alpha coupled via a linker to IL-15, and shown that it displayed far better efficacy than IL-15 in vitro. In this report, we investigated in vivo whether RLI would be a better alternative than IL-15 and IL-2 for cancer treatment using two distinct animal models. B16F10 mouse melanoma cells were injected in C57BL/6 mice either i.v. or intrasplenically for lung or liver metastasis, respectively. HCT-116 human colorectal cancer cells were injected in the cecum of nude mice. We show that RLI has a higher efficiency than IL-15 or IL-2 to reduce lung and liver metastasis and enhance survival in the mouse B16F10 melanoma model, a result that was associated with a higher half-life in vivo. We also found that the antitumoral effect of RLI was completely abolished by in vivo depletion of natural killer cells using anti-asialoGM1 antibody. Moreover, RLI was also efficient to reduce by 50% tumor growth and the progression of metastasis of human colon carcinoma cells in an orthotopic nude mouse model. The fusion protein RLI has revealed strong anticancer effect in two different cancer models overcoming the limited effect of IL-15 by increasing its bioavailability and efficiency. These findings hold significant importance for the use of RLI as a potential adjuvant/therapeutic.

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 histocompatibility 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(-/-)gamma c(-/-) 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 alpha (IL-15R alpha) significantly augmented human NK cells. IL-15-IL-15R alpha 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 CD56(hi)CD16(-)KIR(-) to CD56(lo)CD16(+)KIR(-), and finally to CD56(lo)CD16(+)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.

Defective activations of STAT3 Ser727 and PKC isoforms lead to oncostatin M resistance in metastatic melanoma cells.

Stage III melanoma is refractory to common therapies and shows resistance to the anti-proliferative activity of cytokines in vitro. We previously demonstrated that, for 30% of the metastatic melanoma cell lines, oncostatin M (OSM) resistance is due to the epigenetic silencing of its receptor OSMRbeta. Here we analyse, on a larger panel of short-term cultures derived from melanoma-invaded lymph nodes, other mechanisms potentially implicated in OSM resistance. For 18% of the cell lines, OSM resistance is associated with a phosphorylation defect of signal transducer and activator of transcription (STAT)3 on serine (Ser)727, in concordance with defects in the activation of various protein kinase C (PKC) isoforms, especially PKCdelta. For 21% of the cell lines, OSM resistance is associated with a defect in the activation of Akt on Ser473. By the use of inhibitors, dominant negatives and small interfering (si)RNA, we show that the PKC-STAT3 Ser727, but not the Akt, pathway appears necessary for OSM anti-proliferative activity. Moreover, we bring evidence that OSM or interleukin (IL)-6, produced in lymph nodes and/or melanoma cells, could be involved in the establishment of OSM resistance during melanoma progression. These findings could be relevant for the prognosis and the treatment of stage III melanoma patients.

The exon-3-encoded domain of IL-15ralpha contributes to IL-15 high-affinity binding and is crucial for the IL-15 antagonistic effect of soluble IL-15Ralpha.

We previously showed that a natural soluble form of interleukin-15 (IL-15) Ralpha corresponding to the full-length ectodomain of IL-15Ralpha behaved as a potent antagonist of IL-15 action through IL-15Ralpha/beta/gamma, whereas a recombinant soluble IL-15Ralpha sushi domain did not, but instead acted as an agonist of IL-15 action through IL-15Rbeta/gamma. 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-15Ralpha (sIL-15Ralpha) 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-15Ralpha 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-15Ralpha are agonists on IL-15Rbeta/gamma, 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-15Ralpha in the context of IL-15Ralpha/beta/gamma is due to the 13-aa peptide that creates a sterical constraint impeding the binding of the sIL-15Ralpha.IL-15 complex to the membrane-anchored IL-15Ralpha/beta/gamma. In the frame of the soluble IL-15Ralpha 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-15Ralpha sushi domain-IL-15 fusion protein is therefore able to bind and activate both the IL-15Rbeta/gamma and the IL-15Ralpha/beta/gamma receptors.