Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming (\"Prime\" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 (\"Expand\" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting \"Prime and Expand\" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the \"Prime and Expand\" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

BACKGROUND: Adoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy patients experience a relapse after CAR-T therapy, likely due to inadequate persistence of the modified T cells in the body. IL15, known for its pro-survival and proliferative properties, has been suggested for incorporation into the fourth generation of CAR-T cells to enhance their persistence. However, the potential systemic toxicity associated with this cytokine warrants further evaluation.
METHODS: We analyzed the persistence, antitumor efficacy and potential toxicity of anti-mouse CD19 CAR-T cells which express a membrane-bound IL15-IL15Rα chimeric protein (CD19/mbIL15q CAR-T), in BALB/c mice challenged with A20 tumor cells as well as in NSG mice.
RESULTS: Conventional CD19 CAR-T cells showed low persistence and poor efficacy in BALB/c mice treated with mild lymphodepletion regimens (total body irradiation (TBI) of 1 Gy). CD19/mbIL15q CAR-T exhibits prolonged persistence and enhanced in vivo efficacy, effectively eliminating established A20 B cell lymphoma. However, this CD19/mbIL15q CAR-T displays important long-term toxicities, with marked splenomegaly, weight loss, transaminase elevations, and significant inflammatory findings in some tissues. Mice survival is highly compromised after CD19/mbIL15q CAR-T cell transfer, particularly if a high TBI regimen is applied before CAR-T cell transfer.
CONCLUSIONS: Tethered IL15-IL15Rα augments the antitumor activity of CD19 CAR-T cells but displays long-term toxicity in immunocompetent mice. Inducible systems to regulate IL15-IL15Rα expression could be considered to control this toxicity.

UNASSIGNED: Inflammatory cytokines play key pathogenic roles in liver fibrosis. IL-15 is a proinflammatory cytokine produced by myeloid cells. IL-15 promotes pathogenesis of several chronic inflammatory diseases. However, increased liver fibrosis has been reported in mice lacking IL-15 receptor alpha chain (IL-15Rα), suggesting an anti-fibrogenic role for IL-15. As myeloid cells are key players in liver fibrosis and IL-15 signaling can occur independently of IL-15Rα, we investigated the requirement of IL-15 and IL-15Rα in liver fibrosis.
UNASSIGNED: We induced liver fibrosis in Il15-/- , Il15ra-/- and wildtype C57BL/6 mice by the administration of carbon tetrachloride (CCl4). Liver fibrosis was evaluated by Sirius red and Mason\'s trichrome staining and α-smooth muscle acting immunostaining of myofibroblasts. Gene expression of collagens, matrix modifying enzymes, cytokines and chemokines was quantified by RT-qPCR. The phenotype and the numbers of intrahepatic lymphoid and myeloid cell subsets were evaluated by flow cytometry.
UNASSIGNED: Both Il15-/- and Il15ra-/- mice developed markedly reduced liver fibrosis compared to wildtype control mice, as revealed by reduced collagen deposition and myofibroblast content. Il15ra-/- mice showed further reduction in collagen deposition compared to Il15-/- mice. However, Col1a1 and Col1a3 genes were similarly induced in the fibrotic livers of wildtype, Il15-/- and Il15ra-/- mice, although notable variations were observed in the expression of matrix remodeling enzymes and chemokines. As expected, Il15-/- and Il15ra-/- mice showed markedly reduced numbers of NK cells compared to wildtype mice. They also showed markedly less staining of CD45+ immune cells and CD68+ macrophages, and significantly reduced inflammatory cell infiltration into the liver, with fewer pro-inflammatory and anti-inflammatory monocyte subsets compared to wildtype mice.
UNASSIGNED: Our findings indicate that IL-15 exerts its profibrogenic role in the liver by promoting macrophage activation and that this requires trans-presentation of IL-15 by IL-15Rα.

Metabolic disorders are highly prevalent in modern society. Exercise mimetics are defined as pharmacological compounds that can produce the beneficial effects of fitness. Recently, there has been increased interest in the role of eugenol and transient receptor potential vanilloid 1 (TRPV1) in improving metabolic health. The aim of this study was to investigate whether eugenol acts as an exercise mimetic by activating TRPV1. Here, we showed that eugenol improved endurance capacity, caused the conversion of fast-to-slow muscle fibers, and promoted white fat browning and lipolysis in mice. Mechanistically, eugenol promoted muscle fiber-type transformation by activating TRPV1-mediated CaN signaling pathway. Subsequently, we identified IL-15 as a myokine that is regulated by the CaN/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. Moreover, we found that TRPV1-mediated CaN/NFATc1 signaling, activated by eugenol, controlled IL-15 levels in C2C12 myotubes. Our results suggest that eugenol may act as an exercise mimetic to improve metabolic health via activating the TRPV1-mediated CaN signaling pathway.

Antigen-driven human effector-memory CD8+ T cells expressing low levels of the CD8β chain have been previously described. However, little is known on a possible antigen-independent trigger. We have examined the impact that IL-15 has on the expression of CD8β on purified human naïve CD8+ T cells after CFSE labeling and culture with IL-15. As expected, IL-15 induced naïve CD8+ T cells to proliferate and differentiate. Remarkably, the process was associated with a cell-cycle dependent down-modulation of CD8β from the cell surface, leading to the generation of CD8αβlow and CD8αβ- (i.e., CD8αα) T cells. In contrast, expression of the CD8α chain remained steady or even increased. Neither IL-2 nor IL-7 reproduced the effect of IL-15. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while levels of the M-1/M-2 isoforms and of CD8α increased. Noteworthy, CD8+ T cell blasts obtained after culture of CD8+ T cells with IL-15 showed a cell-cycle dependent increase in the level of the tyrosine kinase Lck, when compared to CD8+ T cells at day 0. This study has shown for the first time that IL-15 generates CD8αα+αβlow and CD8αα+αβ- T cells containing high levels of Lck, suggesting that they may be endowed with unique functional features.

Natural killer (NK) cells play a crucial role in immune response against viral infections and tumors. However, further investigation is needed to better understand the key molecules responsible for determining the fate and function of NK cells. In this study, we made an important discovery regarding the involvement of the Hippo kinases Mst1 and Mst2 as novel regulators in maintaining mouse NK cell homeostasis. The presence of high Mst1 and Mst2 (Mst1/2) activity in NK cells is essential for their proper development, survival and function in a canonical Hippo signaling independent mode. Mechanistically, Mst1/2 induce cellular quiescence by regulating the processes of proliferation and mitochondrial metabolism, thereby ensuring the development and survival of NK cells. Furthermore, Mst1/2 effectively sense IL-15 signaling and facilitate the activation of pSTAT3-TCF1, which contributes to NK cell homeostasis. Overall, our investigation highlights the crucial role of Mst1/2 as key regulators in metabolic reprogramming and transcriptional regulation for mouse NK cell survival and function, emphasizing the significance of cellular quiescence during NK cell development and functional maturation.

Interleukin-7 (IL-7) is considered a critical regulator of memory CD8+ T cell homeostasis, but this is primarily based on analysis of circulating and not tissue-resident memory (TRM) subsets. Furthermore, the cell-intrinsic requirement for IL-7 signaling during memory homeostasis has not been directly tested. Using inducible deletion, we found that Il7ra loss had only a modest effect on persistence of circulating memory and TRM subsets and that IL-7Rα was primarily required for normal basal proliferation. Loss of IL-15 signaling imposed heightened IL-7Rα dependence on memory CD8+ T cells, including TRM populations previously described as IL-15-independent. In the absence of IL-15 signaling, IL-7Rα was upregulated, and loss of IL-7Rα signaling reduced proliferation in response to IL-15, suggesting cross-regulation in memory CD8+ T cells. Thus, across subsets and tissues, IL-7 and IL-15 act in concert to support memory CD8+ T cells, conferring resilience to altered availability of either cytokine.

Chimeric antigen receptor (CAR) T cells have demonstrated outstanding therapeutic success in hematological malignancies. Yet, their efficacy against solid tumors remains constrained due to inadequate infiltration of cytotoxic T and CAR-T cells in the tumor microenvironment (TME), a factor correlated with poor prognosis in patients with solid tumors. To overcome this limitation, we engineered CAR-T cells to secrete CXCL10 and IL15 (10 × 15 CAR-T), which sustain T cell viability and enhance their recruitment, thereby amplifying the long-term cytotoxic capacity of CAR-T cells in vitro. In a xenograft model employing NUGC4-T21 cells, mice receiving 10 × 15 CAR-T cells showed superior tumor reduction and extended survival rates compared to those treated with second-generation CAR-T cells. Histopathological evaluations indicated a pronounced increase in cytotoxic T cell accumulation in the TME post 10 × 15 CAR-T cell treatment. Therefore, the synergistic secretion of CXCL10 and IL15 in these CAR-T cells enhances T cell recruitment and adaptability within tumor tissues, improving tumor control. This approach may offer a promising strategy for advancing CAR-T therapies in the treatment of solid tumors.

The prevention of postoperative recurrence after endoscopic sinus surgery (ESS) relies on targeting specific pathological mechanisms according to individuals\' immunological profiles. However, essential biomarkers and biological characteristics of difficult-to-treat chronic rhinosinusitis (CRS) patients are not well-defined. The aim of this study was to explore the immunologic profiles of subgroups of CRS patients and determine the specific cytokines responsible for recalcitrant or recurrent CRS with nasal polyposis (rCRSwNP). We used 30 cytokine antibody arrays to determine the key cytokines related to recurrent polypogenesis. Enzyme-linked immunosorbent assay (ELISA) experiments were conducted to assess the levels of these key cytokines in 78 patients. Polymorphonuclear leukocytes (PMNs) isolated from nasal polyps were challenged with specific cytokines to examine the levels of enhanced interleukin (IL)-8 production. Finally, we used immunohistochemistry (IHC) staining to check for the presence and distribution of the biomarkers within nasal polyps. A cytokine antibody array revealed that IL-8, IL-13, IL-15, and IL-20 were significantly higher in the recalcitrant CRSwNP group. Subsequent ELISA screening showed a stepwise increase in tissue IL-8 levels in the CHR, CRSsNP, and CRSwNP groups. PMNs isolated from nine CRSwNP cases all demonstrated enhanced IL-8 production after IL-15 treatment. IHC staining was labeled concurrent IL-8 and IL-15 expression in areas of prominent neutrophil infiltration. Our results suggest that IL-15 within the sinonasal mucosa plays a crucial role in promoting IL-8 secretion by infiltrating PMNs in recalcitrant nasal polyps. In addition, we propose a novel therapeutic strategy targeting the anti-IL-15/IL-8 axis to treat CRS with nasal polyposis.

Immunotherapy is widely used in cancer treatment; however, only a subset of patients responds well to it. Significant efforts have been made to identify patients who will benefit from immunotherapy. Successful anti-tumor immunity depends on an intact cancer-immunity cycle, especially long-lasting CD8+ T-cell responses. Interferon (IFN)-α/β/IFN-γ/interleukin (IL)-15 pathways have been reported to be involved in the development of CD8+ T cells. And these pathways may predict responses to immunotherapy. Herein, we aimed to analyze multiple public databases to investigate whether IFN-α/β/IFN-γ/IL-15 pathways could be used to predict the response to immunotherapy. Results showed that IFN-α/β/IFN-γ/IL-15 pathways could efficiently predict immunotherapy response, and guanylate-binding protein 1 (GBP1) could represent the IFN-α/β/IFN-γ/IL-15 pathways. In public and private cohorts, we further demonstrated that GBP1 could efficiently predict the response to immunotherapy. Functionally, GBP1 was mainly expressed in macrophages and strongly correlated with chemokines involved in T-cell migration. Therefore, our study comprehensively investigated the potential role of GBP1 in immunotherapy, which could serve as a novel biomarker for immunotherapy and a target for drug development.