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

ABSTRACT: 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.

SUBMITTER: Bosch NC

PROVIDER: S-EPMC8508776 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Transpresentation of interleukin-15 by IL-15/IL-15Rα mRNA-engineered human dendritic cells boosts antitumoral natural killer cell activity.

Project description:In cancer immunotherapy, the use of dendritic cell (DC)-based vaccination strategies can improve overall survival, but until now durable clinical responses remain scarce. To date, DC vaccines are designed primarily to induce effective T-cell responses, ignoring the antitumor activity potential of natural killer (NK) cells. Aiming to further improve current DC vaccination outcome, we engineered monocyte-derived DC to produce interleukin (IL)-15 and/or IL-15 receptor alpha (IL-15Rα) using mRNA electroporation. The addition of IL-15Rα to the protocol, enabling IL-15 transpresentation to neighboring NK cells, resulted in significantly better NK-cell activation compared to IL-15 alone. Next to upregulation of NK-cell membrane activation markers, IL-15 transpresentation resulted in increased NK-cell secretion of IFN-γ, granzyme B and perforin. Moreover, IL-15-transpresenting DC/NK cell cocultures from both healthy donors and acute myeloid leukemia (AML) patients in remission showed markedly enhanced cytotoxic activity against NK cell sensitive and resistant tumor cells. Blocking IL-15 transpresentation abrogated NK cell-mediated cytotoxicity against tumor cells, pointing to a pivotal role of IL-15 transpresentation by IL-15Rα to exert its NK cell-activating effects. In conclusion, we report an attractive approach to improve antitumoral NK-cell activity in DC-based vaccine strategies through the use of IL-15/IL-15Rα mRNA-engineered designer DC.

| S-EPMC4792546 | biostudies-literature

Adipocytes: A Novel Target for IL-15/IL-15Rα Cancer Gene Therapy.

Project description:IL-15 is a proinflammatory cytokine that plays an essential role in the development and activation of natural killer (NK) cells. Adipose tissue acts as an endocrine organ that secretes cytokines and is an important reservoir for lymphocytes. We hypothesized that activation of the IL-15 signaling in adipose tissue will activate and expand the NK cell population and control tumor growth. We recently developed an adipocyte-targeting recombinant adeno-associated viral (rAAV) vector with minimal off-target transgene expression in the liver. Here, we used this rAAV system to deliver an IL-15/IL-15Rα complex to the abdominal fat by intraperitoneal (i.p.) injection. Adipose IL-15/IL-15Rα complex gene transfer led to the expansion of NK cells in the adipose tissue and spleen in normal mice without notable side effects. The i.p. injection of rAAV-IL-15/IL-15Rα complex significantly suppressed the growth of Lewis lung carcinoma implanted subcutaneously and exerted a significant survival advantage in a B16-F10 melanoma metastasis model. The antitumor effects were associated with the expansion of the NK cells in the blood, spleen, abdominal fat, and tumor, as well as the enhancement of NK cell maturity. Our proof-of-concept preclinical studies demonstrate the safety and efficacy of the adipocyte-specific IL-15/IL-15Rα complex vector as a novel cancer immune gene therapy.

| S-EPMC6520519 | biostudies-literature

Characterization and favorable in vivo properties of heterodimeric soluble IL-15·IL-15Rα cytokine compared to IL-15 monomer.

Project description:Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.

| S-EPMC3689953 | biostudies-literature

IL-15/IL-15Rα Heterodimeric Complex as Cancer Immunotherapy in Murine Breast Cancer Models.

Project description:Interleukin 15 (IL-15) has been evaluated as a potential treatment for solid tumors in clinical trials, but the effectiveness of systemic IL-15 administration as a monotherapy has not been realized. IL-15 receptor alpha (IL-15Rα) can stabilize IL-15 and enhance its bioactivity. The goal of this study was to examine the activity of IL-15/IL-15Rα complex (IL-15cx) to CD8+ T cells and evaluate its potential efficacy in murine breast cancer models. The antitumor efficacy was studied in mouse mammary carcinoma models (Her2/neu transgenic and 4T1-luc mammary cancers) treated with systemic recombinant protein with/without the depletion of myeloid-derived suppressor cells or intra-tumoral gene electrotransfer (GET). IL-15cx shows superior in vivo bioactivity to expand CD8 T cells in comparison to an equimolar single chain IL-15. T-bet is partially involved in CD8 T cell expansion ex vivo and in vivo due to IL-15 or IL-15cx. Intraperitoneal administration of IL-15cx results in a moderate inhibition of breast cancer growth that is associated with an increase in the frequency of cytotoxic CD8 T cells and the improvement of their function. The depletion of myeloid-derived suppressor cells (MDSCs) has no impact on mouse breast cancer growth. IL-15cx treatment diminishes MDSCs in murine tumors. However, it also antagonizes the effects of anti-Gr-1 depleting antibodies. Intratumoral GET with plasmid IL-15/IL-15Rα leads to a long-term survival benefit in 4T1 mammary carcinoma model. An early increase of local cytotoxic cells correlates with GET treatment and an increase of long-term memory T cells results from animals with complete tumor regression. Systemic and local administration of IL-15cx shows two distinct therapeutic responses, a moderate tumor growth inhibition or heterogeneous tumor regressions with survival improvement. Further studies are warranted to improve the efficacy of IL-15cx as an immunotherapy for breast cancer.

| S-EPMC7897681 | biostudies-literature

<i>Trans</i>-endocytosis of intact IL-15Rα-IL-15 complex from presenting cells into NK cells favors signaling for proliferation.

Project description:Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the β and common γ (γc) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα-IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα-IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα-IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα-IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor-ligand interaction occurs can influence functional outcome.

| S-EPMC6955382 | biostudies-literature

IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Rα production.

Project description:Ischemia and reperfusion (I/R) injury following liver transplantation (LTx) is an important problem that significantly impacts clinical outcomes. IFN regulatory factor-1 (IRF-1) is a nuclear transcription factor that plays a critical role in liver injury. Our objective was to determine the immunomodulatory role of IRF-1 during I/R injury following allogeneic LTx. IRF-1 was induced in liver grafts immediately after reperfusion in both human and mouse LTx. IRF-1 contributed significantly to I/R injury because IRF-1-knockout (KO) grafts displayed much less damage as assessed by serum alanine aminotransferase and histology. In vitro, IRF-1 regulated both constitutive and induced expression of IL-15, as well as IL-15Rα mRNA expression in murine hepatocytes and liver dendritic cells. Specific knockdown of IRF-1 in human primary hepatocytes gave similar results. In addition, we identified hepatocytes as the major producer of soluble IL-15/IL-15Rα complexes in the liver. IRF-1-KO livers had significantly reduced NK, NKT, and CD8(+) T cell numbers, whereas rIL-15/IL-15Rα restored these immune cells, augmented cytotoxic effector molecules, promoted systemic inflammatory responses, and exacerbated liver injury in IRF-1-KO graft recipients. These results indicate that IRF-1 promotes LTx I/R injury via hepatocyte IL-15/IL-15Rα production and suggest that targeting IRF-1 and IL-15/IL-15Rα may be effective in reducing I/R injury associated with LTx.

| S-EPMC4458432 | biostudies-literature

Virally infected and matured human dendritic cells activate natural killer cells via cooperative activity of plasma membrane-bound TNF and IL-15.

Project description:Recombinant adenovirus-engineered dendritic cells (Ad.DCs) are potent immunologic adjuvants of antiviral and anticancer vaccines. The effectiveness of Ad.DC-based vaccines may depend on the ability of Ad.DCs to crosstalk with natural killer (NK) cells and to activate, polarize, and bridge innate and adaptive immunity. We investigated, for the first time, whether and how human Ad.DCs activate NK cells, and compared the Ad.DC function with that of immature DCs and matured DCs (mDCs). We found that adenovirus transduction and lipopolysaccharide/interferon-gamma-induced maturation increased expression of transmembrane tumor necrosis factor (TNF) and trans-presented (trans) interleukin-15 (IL-15) on DCs, leading to enhanced NK cell activation without enhancing DC susceptibility to NK cell-mediated killing. This crosstalk enhanced NK cell CD69 expression, interferon-gamma secretion, proliferation, and antitumor activities, with Ad.DCs being significantly more effective than immature DCs, but less effective than mDCs. The Ad.DC and mDC crosstalk with NK cells was largely prevented by physical separation of DCs and NK cells, and neutralization of total TNF and IL-15, but not by selective sequestration of soluble TNF. These findings demonstrate that both Ad.DCs and mDCs can efficiently promote innate immune functions by activation of NK cells through the cooperative activities of tmTNF and trans-IL-15 mediated by cell-to-cell contact.

| S-EPMC3324291 | biostudies-literature

Development of an IL-15-autocrine CD8 T-cell leukemia in IL-15-transgenic mice requires the cis expression of IL-15Rα.

Project description:IL-15 has growth-promoting effects on select lymphoid subsets, including natural killer (NK) cells, NK T cells, intraepithelial lymphocytes (IELs), CD8 T cells, and γδ-T cells. Constitutive expression of murine IL-15 in IL-15-transgenic mice was reported to cause T-NK leukemia. We investigated whether IL-15 expression is sufficient for leukemic transformation using a human IL-15-transgenic (IL-15Tg) mouse model. We noted that 100% of the mice observed over a 2-year period (n > 150) developed fatal expansions of CD8 T cells with NK markers, and determined that these cells expressed IL-15 receptor alpha (IL-15Rα). The expression of IL-15Rα on CD8 T cells appears to be required for uncontrolled aggressive lymphoproliferation, because none of the IL-15Rα(-/-)-IL-15Tg mice that we followed for more than 2 years developed the fatal disease despite controlled expansion of CD8 T cells. In addition, in contrast to IL-15Tg mice, in which leukemia-like CD8 T cells expressed IL-15Rα persistently, acutely activated normal CD8 T cells only transiently expressed IL-15Rα. Inhibition of DNA methylation enabled sustained IL-15Rα expression induced by activation. We present a scenario for IL-15Tg mice in which CD8 T cells that acquire constitutive persistent IL-15Rα expression are at a selective advantage and become founder cells, outgrow other lymphocytes, and lead to the establishment of a leukemia-like condition.

| S-EPMC3087530 | biostudies-literature

IL-27 enhances IL-15/IL-18-mediated activation of human natural killer cells.

Project description:BackgroundNatural killer (NK) cells are an emerging new tool for cancer immunotherapy. To develop NK cell therapeutics from peripheral blood mononuclear cells (PBMCs) of healthy donors, substantial expansion of primary NK cells is necessary because of the very low number of these cells in peripheral blood. In this study, we aimed to investigate the effect of various cytokine alone or combinations, in expanded NK cells and to analyze the synergetic effect of cytokine combinations.MethodsHuman NK cells were isolated from healthy donor PBMC. Purified NK cells were stimulated with single cytokines or combinations of IL-2, IL-15, IL-18, and IL-27. The expanded NK cells were characterized by flow cytometry, cytotoxicity assay, calcein AM assay and Western blot.ResultsWe investigated the synergistic effects of each cytokine, namely, IL-2, IL-15, IL-18, and IL-27, on human NK cells isolated from PBMCs of healthy donors and cultured for 21 days. We identified that IL-15/IL-18/IL-27-mediated activation of NK cells most potently increased NK cell proliferation, cytotoxicity, and IFN-ɣ secretion compared with the activation observed with other treatments, including IL-2, IL-15, and IL-15/IL-18. Additionally, the expression of DNAM-1, NKG2D, CD69, and natural cytotoxicity receptors (NCRs; NKp30 and NKp44) increased on day 21 compared to that on day 0, demonstrating the activation of NK cells. In vitro, expanded NK cells were highly cytotoxic against cancer cells, displaying increased perforin and granzyme B accumulation.ConclusionsTaken together, these results indicated that IL-27 can synergize on NK cell expansion and activation with IL-15 and IL-18. In addition, we described an improved culture method for ex vivo expansion of human NK cells with IL-15/IL-18/IL-27 stimulation and characterized the response of NK cells to this stimulation.

| S-EPMC6612093 | biostudies-literature

Paracrine and transpresentation functions of IL-15 are mediated by diverse splice versions of IL-15Rα in human monocytes and dendritic cells.

Project description:BackgroundIL-15 can either be transpresented by IL-15Rα or be secreted.ResultsNew N- and C-terminal splice versions of human IL-15Rα determine whether IL-15 is secreted or stays bound to the cell membrane.ConclusionIL-15Rα isoforms determine the mode of action of IL-15.SignificanceIL-15Rα isoforms may modify immune response outcomes in humans. Species-specific differences of post-translational modifications suggested the existence of human IL-15Rα isoforms. We identified eight new isoforms that are predicted to modify the intracellular C termini of IL-15Rα, and another N-terminal exon "Ex2A" that was consistently present in all but one of the C-terminal isoforms. Ex2A encodes a 49-amino acid domain that allowed the transfer of IL-15/IL-15Rα complex to the cell surface but prevented its cleavage from cell membranes and its secretion thus facilitating the transpresentation of IL-15 as part of the immunological synapse. The Ex2A domain also affected the O-glycosylation of IL-15Rα that explained the species-specific differences. The Ex2A domain appeared to be removed from major IL-15Rα species during protein maturation, but both Ex2A and IL-15Rα appeared on the surface of monocytic cells upon activation. The membrane-associated form of the only C-terminal isoform that lacked Ex2A (IC3) was retained inside the cell, but soluble IL-15/IL-15Rα complexes were readily released from cells that expressed IL-15/IL-15Rα-IC3 thus limiting this IL-15/IL-15Rα isoform to act as a secreted molecule. These data suggest that splice versions of IL-15Rα determine the range of IL-15 activities.

| S-EPMC3504748 | biostudies-literature

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