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Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses but the factors driving NK cell IL-10 secretion are poorly-defined. Here, we identified NK cell-intrinsic STAT3 activation as vital for IL-10 production during both systemic (Lm) infection and following IL-15 cytokine/receptor complex (IL15C) treatment for experimental cerebral malaria (ECM). In both contexts, conditional deficiency in NK cells abrogated production of IL-10. Initial NK cell STAT3 phosphorylation was driven by IL-15. During Lm infection, this required capture or presentation of IL-15 by NK cell IL-15Rα. Persistent STAT3 activation was required to drive measurable IL-10 secretion and required NK cell expression of IL-10Rα. Survival-promoting effects of IL-15C treatment in ECM were dependent on NK cell while NK cell-intrinsic deficiency for , or abrogated NK cell IL-10 production and increased resistance against Lm. NK cell deficiency did not impact production of IFNγ, indicating the STAT3 activation initiated by IL-15 and amplified by IL-10 selectively drives the production of anti-inflammatory IL-10 by responding NK cells.

Rapid advancements in understanding how the immune system can eliminate tumors have quickly translated into breakthroughs in developing cancer therapeutics. Immune checkpoint inhibitors (ICIs) have shown great promise in several cancers; however, resistance can affect up to two-thirds of patients receiving ICIs. A significant limitation of the effectiveness of anti-PD-1 therapy centers around the insufficient levels of immune cells needed to recognize and kill cancer cells compared to the number of suppressive immune cells within the tumor microenvironment. Determining what is required to overcome the resistance to anti-PD-1 therapy in breast cancer remains a critical need. Our data demonstrate that IL-15 complexes injected intratumorally in combination with PD-1 blockade therapy induce regression of established luminal B mammary breast tumors. We show that IL-15 alone or in combination with anti-PD-1 drives changes in gene expression of pathways associated with TCR and co-stimulatory signaling, immune cell adhesion, and migration. Furthermore, we show that intratumoral injection of IL-15 complexes traffics to the tumor-draining lymph node, as evidenced by Light sheet microscopy, and colocalizes with the anti-PD-1 monoclonal antibody. We also identify the immune signatures, localization, and distribution of immune cells in regressing and non-regressing breast tumors.

BackgroundAs 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.MethodsWe 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).ResultsRLI 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.ConclusionsCollectively, 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.

Invariant natural killer T (iNKT) cells are a distinct subpopulation of innate-like T lymphocytes. They are characterized by semi-invariant T cell receptors (TCRs) that recognize both self and foreign lipid antigens presented by CD1d, a non-polymorphic MHC class I-like molecule. iNKT cells play a critical role in stimulating innate and adaptive immune responses, providing an effective defense against infections and cancers, while also contributing to chronic inflammation. The functions of iNKT cells are specific to their location, ranging from lymphoid to non-lymphoid tissues, such as the thymus, lung, liver, intestine, and adipose tissue. This review aims to provide insights into the heterogeneity of development and function in iNKT cells. First, we will review the expression of master transcription factors that define subsets of iNKT cells and their production of effector molecules such as cytokines and granzymes. In this article, we describe the gene expression profiles contributing to the kinetics, distribution, and cytotoxicity of iNKT cells across different tissue types. We also review the impact of cytokine production in distinct immune microenvironments on iNKT cell heterogeneity, highlighting a recently identified circulating iNKT cell subset. Additionally, we explore the potential of exploiting iNKT cell heterogeneity to create potent immunotherapies for human cancers in the future.

Background Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by an immunosuppressive microenvironment. To address the immune desert in GBM, immunocytokine, an innovative recombinant fusion protein that combines antibodies and cytokines, has been proposed. This molecule, targeting tumor cells while stimulating immune cells, effectively enhances the anti-tumor response. Developing a novel immunocytokine presents a promising strategy for GBM. Methods In this study, we developed a novel immunocytokine, αBC-IL15, composed of a bispecific T cell engager (BsTCE) fused to the interleukin-15 receptor alpha-sushi domain/interleukin-15 complex (IL-15/IL-15Rα). To analyze its affection on immune cells, we co-cultured it with peripheral blood mononuclear cells in vitro. To investigate whether it enhances the cytotoxicity of T cells compared to B7-H3 BsTCE, we performed cytotoxicity assays on cell lines and spheroids in vitro. Moreover, we assessed its efficacy in vivo and analyzed the tumor microenvironment using flow cytometry and bulk sequencing. Results Our study assessed the bioactivity and efficiency of αBC-IL15. In vitro, αBC-IL15 effectively bound to tumor cells and T cells, inducing immune cell activation, cytokine release, and cytotoxicity toward tumor cells. In vivo, αBC-IL15 connects cytotoxic immune cells with tumor cells, leading to effective immune cell infiltration and potent redirected lysis. Conclusions Overall, we developed a novel bispecific immunocytokine, αBC-IL15, which targets tumor cells while enriching and activating effector immune cells. This agent significantly enhanced anti-tumor effects both in vitro and in vivo, highlighting its potential to overcome the immunosuppressive microenvironment in GBM. Graphical Abstract

While autologous CAR T-cell therapies have revolutionized the treatment of hematologic malignancies, their widespread application is hindered by manufacturing complexities, high costs, and limited efficacy against solid tumors due to antigen heterogeneity and the TME. Moreover, the logistical burden of bespoke patient-specific manufacturing restricts global scalability. In response, the immunotherapy landscape is pivoting toward “off-the-shelf” allogeneic therapies derived from innate and innate-like effectors. This review provides a comprehensive analysis of four emerging platforms: CAR-NK cells, CAR-NKT cells, γδ T cells, and CAR-M. Unlike conventional αβ T cells, these lineages utilize MHC-independent mechanisms to recognize stress-induced ligands or lipid antigens, inherently minimizing the risk of GvHD while enabling standardized, batched manufacturing. We critically examine the diverse manufacturing paradigms, contrasting the scalability of iPSC-derived sources with the accessibility of umbilical cord blood products. Furthermore, we detail advanced engineering strategies designed to overcome the lineage-specific limitations revealed by early trials—specifically, “armoring” constructs with IL-15 to boost in vivo persistence and metabolic reprogramming to sustain function within the TME. Finally, we synthesize emerging clinical evidence which confirms the favorable safety profile of these allogeneic approaches but highlights persistent bottlenecks: limited durability of response, cryopreservation-induced loss of viability, and batch-to-batch variability. We conclude that unlocking the full potential of innate CAR therapies requires a dual focus on harmonizing manufacturing controls and developing next-generation engineering logic to ensure durable control of solid tumors.

Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both and by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15 tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5 and NKp46 (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

Celiac disease is a T cell-mediated immune disorder induced by dietary gluten that is characterized by the development of an inflammatory anti-gluten CD4 T cell response, anti-gluten antibodies, and autoantibodies against tissue transglutaminase 2 and the activation of intraepithelial lymphocytes (IELs) leading to the destruction of the intestinal epithelium. Intraepithelial lymphocytes represent a heterogeneous population of T cells composed mainly of cytotoxic CD8 T cells residing within the epithelial layer, whose main role is to maintain the integrity of the epithelium by eliminating infected cells and promoting epithelial repair. Dysregulated activation of IELs is a hallmark of CD and is critically involved in epithelial cell destruction and the subsequent development of villous atrophy. In this review, we compare and contrast the phenotype and function of human and mouse small intestinal IELs under physiological conditions. Furthermore, we discuss how conditions of epithelial distress associated with overexpression of IL-15 and non-classical MHC class I molecules induce cytotoxic IELs to become licensed killer cells that upregulate activating NKG2D and CD94/NKG2C natural killer receptors, acquiring lymphokine killer activity. Pathways leading to dysregulated IEL activation could eventually be targeted to prevent villous atrophy and treat patients who respond poorly to gluten-free diet.

Studies to develop cell-based therapies for cancer and other diseases have consistently shown that purified human natural killer (NK) cells secrete cytokines and kill target cells after in vitro culture with high concentrations of cytokines. However, these assays poorly reflect the conditions that are likely to prevail in vivo in the early stages of an infection and have been carried out in a wide variety of experimental systems, which has led to contradictions within the literature. We have conducted a detailed kinetic and dose-response analysis of human NK cell responses to low concentrations of IL-12, IL-15, IL-18, IL-21, and IFN-α, alone and in combination, and their potential to synergize with IL-2. We find that very low concentrations of both innate and adaptive common γ chain cytokines synergize with equally low concentrations of IL-18 to drive rapid and potent NK cell CD25 and IFN-γ expression; IL-18 and IL-2 reciprocally sustain CD25 and IL-18Rα expression in a positive feedback loop; and IL-18 synergizes with FcγRIII (CD16) signaling to augment antibody-dependent cellular cytotoxicity. These data indicate that NK cells can be rapidly activated by very low doses of innate cytokines and that the common γ chain cytokines have overlapping but distinct functions in combination with IL-18. Importantly, synergy between multiple signaling pathways leading to rapid NK cell activation at very low cytokine concentrations has been overlooked in prior studies focusing on single cytokines or simple combinations. Moreover, although the precise common γ chain cytokines available during primary and secondary infections may differ, their synergy with both IL-18 and antigen-antibody immune complexes underscores their contribution to NK cell activation during innate and adaptive responses. IL-18 signaling potentiates NK cell effector function during innate and adaptive immune responses by synergy with IL-2, IL-15, and IL-21 and immune complexes.

Interleukin （IL）-15 plays an important role in natural killer （NK） and CD8＋ T-cell proliferation and function and is more effective than IL-2 for tumor immunotherapy. The trans-presentation of IL-15 by neighboring cells is more effective for NK cell activation than its soluble IL-15. In this study, the fusion protein dsNKG2D-IL-15, which consisted of two identical extracellular domains of human NKG2D coupled to human IL-15 via a linker, was engineered in Escherichia coll. DsNKG2D-IL- 15 could efficiently bind to major histocompatibility complex class I chain-related protein A （MICA） of human tumor cells with the two NKG2D domains and trans-present IL-15 to NK or CD8＋ T cells. We transplanted human gastric cancer （SGC-7901） cells into nude mice and mouse melanoma cells with ectopic expression of MICA （B 16BL6-MICA） into C57BL/6 mice. Then, we studied the anti-tumor effects mediated by dsNKG2D-IL-15 in the two xenografted tumor models. Human dsNKG2D-IL-15 exhibited higher efficiency than IL-15 in suppressing gastric cancer growth. Exogenous human dsNKG2D-IL-15 was centrally distributed in the mouse tumor tissues based on in vivo live imaging. The frequencies of human CD56＋ cells infiltrated into the tumor tissues following the injection of peripheral blood mononuclear cells into nude mice bearing human gastric cancer were significantly increased by human dsNKG2D-I L- 15 treatment. Human dsNKG2 D-I L- 15 also delayed the growth of transplanted melanoma （B 16BL6- MICA） by activating and recruiting mouse NK and CD8＋ T cells. The anti-melanoma effect of human dsNKG2D-IL-15 in C57BL/6 mice was mostly decreased by the in vivo depletion of mouse NK cells. These data highlight the potential use of human dsNKG2D-IL-15 for tumor therapy.