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Background Chimeric antigen receptor (CAR)-T cell therapy is a powerful adoptive immunotherapy against both B-cell malignancies and some types of solid tumors. Interleukin (IL) -15 is an important immune stimulator that may provide ideal long-term persistent CAR-T cells. However, higher base line or peak serum IL-15 levels are also related to severe toxicity, such as cytokine release syndrome (CRS), graft-versus-host disease (GVHD), and neurotoxicity. Methods We successfully constructed CD19 specific armored CAR-T cells overexpressing IL-I5 and IL-15 receptor alpha (IL-15Ra). In vitro cell differentiation and viability were monitored by flow cytometry, and an in vivo xenograft mouse models was used to evaluate the anti-tumor efficiency and liver damage of CAR-T cells. Results CAR-T cells overexpressing IL-15 alone demonstrated enhanced viability, retarded exhaustion in vitro and superior tumor-inhibitory effects in vivo. However, these tumor-free mice had lower survival rates, with serious liver injuries, as a possible result of toxicity. As expected, CAR-T cells overexpressing IL-15 combined with IL-15Ra had reduced CD132 expression and released fewer cytokines (IFNγ, IL-2 and IL-15) in vitro, as well as had the tendency to improve mouse survival via repressing the growth of tumor cells and keeping livers healthier compared to CAR-IL-15 T cells. Conclusions These results indicated the importance of IL-15 in enhancing T cells persistence and IL-15Ra in reducing the adverse effects of IL-15, with superior tumor retardation during CAR-T therapy. This study paves the way for the rapid exploitation of IL-15 in adoptive cell therapy in the future.

Cytokine therapy, involving interleukin-15 (IL-15), is a promising strategy for cancer immunotherapy. However, clinical application has been limited due to severe toxicity and the relatively low immune response rate, caused by wide distribution of cytokine receptors, systemic immune activation and short half-life of IL-15. Here we show that a biomimetic nanovaccine, developed to co-deliver IL-15 and an antigen/major histocompatibility complex (MHC) selectively targets IL-15 to antigen-specific cytotoxic T lymphocytes (CTL), thereby reducing off-target toxicity. The biomimetic nanovaccine is composed of cytomembrane vesicles, derived from genetically engineered dendritic cells (DC), onto which IL-15/IL-15 receptor α (IL-15Rα), tumor-associated antigenic (TAA) peptide/MHC-I, and relevant costimulatory molecules are simultaneously anchored. We demonstrate that, in contrast to conventional IL-15 therapy, the biomimetic nanovaccine with multivalent IL-15 self-transpresentation (biNV-IL-15) prolonged blood circulation of the cytokine with an 8.2-fold longer half-life than free IL-15 and improved the therapeutic window. This dual targeting strategy allows for spatiotemporal manipulation of therapeutic T cells, elicits broad spectrum antigen-specific T cell responses, and promotes cures in multiple syngeneic tumor models with minimal systemic side effects. Cancer immune therapy utilizing interleukin 15 (Il-15) is hampered by the short half-life and systemic toxic effects of the cytokine. Here authors introduce a biomimetic nanovaccine, in which Il-15 and tumor-associated antigenic peptide/MHC-I complexes are co-anchored to cell membrane vesicles of dendritic cell origin, which elicits antigen-specific T cell response leading to superior anti-tumour effect in syngeneic mouse tumour models.

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.

Natural killer (NK) cells were so named for their uniqueness in killing certain tumor and virus-infected cells without prior sensitization. Their functions are modulated in vivo by several soluble immune mediators; interleukin-15 (IL-15) being the most potent among them in enabling NK cell homeostasis, maturation, and activation. During microbial infections, NK cells stimulated with IL-15 display enhanced cytokine responses. This priming effect has previously been shown with respect to increased IFN-γ production in NK cells upon IL-12 and IL-15/IL-2 co-stimulation. In this study, we explored if this effect of IL-15 priming can be extended to various other cytokines and observed enhanced NK cell responses to stimulation with IL-4, IL-21, IFN-α, and IL-2 in addition to IL-12. Notably, we also observed elevated IFN-γ production in primed NK cells upon stimulation through the Ly49H activation receptor. Currently, the fundamental processes required for priming and whether these signaling pathways work collaboratively or independently for NK cell functions are poorly understood. To identify the key signaling events for NK cell priming, we examined IL-15 effects on NK cells in which the pathways emanating from IL-15 receptor activation were blocked with specific inhibitors. Our results demonstrate that the PI3K-AKT-mTOR pathway is critical for cytokine responses in IL-15 primed NK cells. Furthermore, this pathway is also implicated in a broad range of IL-15-induced NK cell effector functions such as proliferation and cytotoxicity. Likewise, NK cells from mice treated with rapamycin to block the mTOR pathway displayed defects in proliferation, and IFN-γ and granzyme B productions resulting in elevated viral burdens upon murine cytomegalovirus infection. Taken together, our data demonstrate the requirement of PI3K-mTOR pathway for enhanced NK cell functions by IL-15, thereby coupling the metabolic sensor mTOR to NK cell anti-viral responses.

Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor β chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1β induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1β to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1βR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation. Mucosal-associated invariant T (MAIT) cells are key in immunity and diseases, but how their effector polarization is controlled is still unclear. Here, the authors show that an IL-1β/IL-23/mTORC2 axis is essential for the induction of IL-17-producing MAIT17, while an IL-2/IL-15/mTORC1 axis is important for the homeostasis of IFN-γ-producing MAIT1.

Interleukin‐15 (IL‐15), a pro‐inflammatory cytokine, is produced mainly by skeletal muscle cells, macrophages and epithelial cells. Recent research has demonstrated that IL‐15 is closely related to the functions of bone and skeletal muscle in the locomotor system. There is growing evidence that exercise, an important means to regulate the immune and locomotor systems, influences IL‐15 content in various tissues, thereby indirectly affecting the function of bones and muscles. Furthermore, the form, intensity, and duration of exercise determine the degree of change in IL‐15 and downstream effects. This paper reviews the structure, synthesis and secretion of IL‐15, the role of IL‐15 in regulating the metabolism of bone tissue cells and myofibers through binding to the IL‐15 receptor‐α (IL‐15Rα), and the response of IL‐15 to different types of exercise. This review provides a reference for further analyses of the role and mechanism of action of IL‐15 in the regulation of metabolism during exercise.

BackgroundIndividuals testing positive for anti-cyclic-citrullinated-peptide-antibodies (Anti-CCP) and musculoskeletal (MSK) complaints are at risk for developing rheumatoid arthritis (RA).ObjectivesWe aim to identify factors involved in arthritis progression in a population considered at risk for RA.MethodsAnti-CCP-positive individuals with MSK complaints referred to rheumatologist in the Region Stockholm were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥3 years or until arthritis diagnosis was made. Blood samples from inclusion were analyzed for 9 selected anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); as well as a panel of 92 inflammation-associated proteins and HLA-SE alleles. Cox regression was applied to the data and a predictive multivariate model was identified. Results are shown with a confidence interval (CI) of 95 percent.Results267 individuals were recruited. 101 (38%) developed arthritis in median after 14 months (IQR: 6-27). In the multivariate analysis: ACPA reactivity (HR 8.0, CI 2.9-22, p<0.0001), IL15R-α levels (HR 0.6, CI 0.4-0.9, p 0.006), IL6 levels (HR 1.5, CI 1.2-1.8, p<0.0001) and the presence of tenosynovitis as detected by ultrasound (HR 3.4, CI 2.0-6.0, p<0.0001) were significantly associated with arthritis. Diagnostic accuracy for ACPA reactivity test had a sensitivity of 96% (CI 92-99.8), a specificity of 30% (CI 30-46), a positive predictive value of 51% (CI 43-58) and a negative predictive value of 94% (CI 87-99.7). Diagnostic accuracy for ultrasound assessed tenosynovitis had a sensitivity of 17% (CI 9-24), a specificity of 99% (CI 97-100), a positive predictive value of 89% (CI 74-100) and a negative predictive value of 64% (CI 57-70).ConclusionWe propose a high-risk RA phase characterized by the presence of certain ACPA reactivities, IL15-Rα, IL6, and tenosynovitis, parameters that could be used to identify individuals at particular low risk and high risk for arthritis progression.AcknowledgementsNone.Disclosure of InterestsAlexandra Cîrciumaru: None declared, Yogan Kisten: None declared, Monika Hansson: None declared, Linda Mathsson-Alm Employee of: ThermoFisher Scientific, Vijay Joshua: None declared, Heidi Wähämaa: None declared, Malena Loberg Haarhaus: None declared, Joakim Lindqvist: None declared, Fei Guozhong: None declared, Nancy Vivar Pomiano: None declared, Hamed Rezaei: None declared, Erik af Klint: None declared, Aleksandra Antovic: None declared, Bence Réthi: None declared, Anca Catrina: None declared, Aase Hensvold: None declared.

Interindividual immune variability is driven predominantly by environmental factors, including exposure to chronic infectious agents such as cytomegalovirus (CMV). We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells-and marked expansion of innate-memory CD8+ T cells. These cells express high levels of NKG2A/C and the IL-2 and IL-15 receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C+CD122+CD8+ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I-deficient K562 targets and prompt IFN-y production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1-vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C+CD8+ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMVvectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innatememory expansion could be achieved by other vaccine platforms expressing IL-15.

Among numerous cytokines modulating natural killer (NK) cell function, interleukin 15 (IL-15) exerts a broad range of effect from development and homeostasis, to activation of mature NK cells during infection. Its significance is further highlighted by clinical trials in which IL-15 is being used to boost the proliferation and anti-tumor response of NK cells. Among the signal transduction pathways triggered by the engagement of IL-15 receptor with its ligand, the PI3K-AKT-mTOR pathway seems to be critical for the IL-15-mediated activation of NK cells, therefore being responsible for efficient anti-viral and anti-tumor responses. This review provides an overview of the role of IL-15 at multiple stages of NK cell life journey. Understanding the pathway by which IL-15 conveys critical signals for the generation of NK cells with efficient effector functions, in combination with established protocols for NK cell expansion ex vivo, will undoubtedly open new avenues for therapeutic applications for immunomodulation against infections and cancers.

The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches.