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Sarcoma IL-12 overexpression facilitates NK cell immunomodulation.

ABSTRACT: Interleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

SUBMITTER: Rademacher MJ

PROVIDER: S-EPMC8050085 | biostudies-literature |

REPOSITORIES: biostudies-literature

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IL-15 Promotes Polyfunctional NK Cell Responses to Influenza by Boosting IL-12 Production.

Project description:IL-15 is a key regulator of NK cell maintenance and proliferation and synergizes with other myeloid cell-derived cytokines to enhance NK cell effector function. At low concentrations, trans-presentation of IL-15 by dendritic cells can activate NK cells, whereas at higher concentrations it can act directly on NK cells, independently of accessory cells. In this study, we investigate the potential for IL-15 to boost responses to influenza virus by promoting accessory cell function. We find that coculture of human PBMCs with inactivated whole influenza virus (A/Victoria/361/2011) in the presence of very low concentrations of IL-15 results in increased production of myeloid cell-derived cytokines, including IL-12, IFN-α2, GM-CSF, and IL-1β, and an increased frequency of polyfunctional NK cells (defined by the expression of two or more of CD107a, IFN-γ, and CD25). Neutralization experiments demonstrate that IL-15-mediated enhancement of NK cell responses is primarily dependent on IL-12 and partially dependent on IFN-αβR1 signaling. Critically, IL-15 boosted the production of IL-12 in influenza-stimulated blood myeloid dendritic cells. IL-15 costimulation also restored the ability of less-differentiated NK cells from human CMV-seropositive individuals to respond to influenza virus. These data suggest that very low concentrations of IL-15 play an important role in boosting accessory cell function to support NK cell effector functions.

| S-EPMC5890538 | biostudies-literature

IL-12-producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion.

Project description:Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C(+) subset and general NK cell recovery rely on signals derived from CD14(+) monocytes. In a coculture system, a subset of CD14(+) cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C(+) subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C(+) NK cells. Together, our results reveal that IL-12, CD14(+) cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C(+) NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C(+) NK cell subset have the potential to be exploited in NK cell-based intervention strategies against viral infections and cancer.

| S-EPMC4348979 | biostudies-literature

IL-12/15/18-induced cell death and mitochondrial dynamics of human NK cells.

Project description:Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memory-like (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy.

| S-EPMC10413107 | biostudies-literature

Prospects for NK Cell Therapy of Sarcoma.

Project description:Natural killer (NK) cells are innate lymphoid cells with potent antitumor activity. One of the most NK cell cytotoxicity-sensitive tumor types is sarcoma, an aggressive mesenchyme-derived neoplasm. While a combination of radical surgery and radio- and chemotherapy can successfully control local disease, patients with advanced sarcomas remain refractory to current treatment regimens, calling for novel therapeutic strategies. There is accumulating evidence for NK cell-mediated immunosurveillance of sarcoma cells during all stages of the disease, highlighting the potential of using NK cells as a therapeutic tool. However, sarcomas display multiple immunoevasion mechanisms that can suppress NK cell function leading to an uncontrolled tumor outgrowth. Here, we review the current evidence for NK cells' role in immune surveillance of sarcoma during disease initiation, promotion, progression, and metastasis, as well as the molecular mechanisms behind sarcoma-mediated NK cell suppression. Further, we apply this basic understanding of NK-sarcoma crosstalk in order to identify and summarize the most promising candidates for NK cell-based sarcoma immunotherapy.

| S-EPMC7763692 | biostudies-literature

IL-18, but not IL-15, contributes to the IL-12-dependent induction of NK-cell effector functions by Leishmania infantum in vivo.

Project description:Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK-cell response in mice that required TLR9-positive myeloid DC and IL-12, but no IFN-alpha/beta. Here, we investigated whether IL-15 or IL-18 mediate the activity of IL-12 or function as independent activators of NK cells. In contrast to earlier studies that described IL-15 as crucial for NK-cell priming in response to TLR ligands, the expression of IFN-gamma, FasL, perforin and granzyme B by NK cells in L. infantum-infected mice was completely preserved in the absence of IL-15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN-gamma secretion, cytotoxicity and FasL expression of NK cells from infected IL-18(-/-) mice were significantly reduced compared with controls, but, unlike IL-12, IL-18 was not essential for NK-cell effector functions. Part of the NK-cell-stimulatory effect of IL-12 was dependent on IL-18. We conclude that IL-15 is not functioning as a universal NK-cell priming signal and that IL-18 contributes to the NK-cell response in visceral leishmaniasis. The cytokine requirements for NK-cell activation appear to differ contingent upon the infectious pathogen.

| S-EPMC2909391 | biostudies-literature

IκBζ augments IL-12- and IL-18-mediated IFN-γ production in human NK cells.

Project description:Interferon-γ (IFN-γ) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of κB-ζ (IκBζ), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-γ production in KG-1 cells, we tested IκBζ's role in the classic lymphocyte pathway of IL-12/IL-18-induced IFN-γ. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of IκBζ and released IFN-γ. CD56(+) NK cells were shown to be the IκBζ-producing lymphocyte subpopulation, which also released abundant IFN-γ in response to IL-12/IL-18. Importantly, IκBζ was undetectable in CD56(-) lymphocytes where IFN-γ release was 10-fold lower. In addition, small interfering RNA knockdown of IκBζ suppressed IFN-γ expression in CD56(+) cells. The association of IκBζ with the IFN-γ promoter was documented by chromatin immunoprecipitation. IFN-γ promoter activity from IκBζ overexpression was confirmed by luciferase reporter assay. Finally, IκBζ coprecipitated with p65 and p50 NF-κB in NK cells in response to IL-12/IL-18, suggesting that IκBζ's effects on IFN-γ promoter activity are coregulated by NF-κB. These results suggest that IκBζ functions as an important regulator of IFN-γ in human NK cells, further expanding the class of IκBζ-modulated genes.

| S-EPMC3062297 | biostudies-literature

Comparison of characteristics and tumor targeting properties of extracellular vesicles derived from primary NK cells or NK-cell lines stimulated with IL-15 or IL-12/15/18.

Project description:NK cell-based therapies have shown promise for hematological cancer forms, but their use against solid tumors is hampered by their poor ability to infiltrate the tumor. NK cells release extracellular vesicles (EVs) containing cytolytic proteins, indicating that NK-cell derived EVs may have therapeutic potential. In this study, we compared the tumor-targeting potential of EVs derived from either primary NK cells or the NK cell lines NK-92 and KHYG-1 cultured in IL-15 alone or in combination with IL-12 and IL-18. Primary NK cells were also stimulated through the activating receptor CD16. Tumor cell apoptosis was measured using a panel of human colon, melanoma, glioblastoma, prostate, breast, and ovarian tumor cell line spheroids. NK cells or NK-92 cells stimulated with IL-12, IL-15, and IL-18 generated EVs with higher efficiency than EVs from resting cells, although similar amounts of EVs were produced under both conditions. Proteomic analysis indicated similar distribution of cytolytic proteins in EVs from primary NK cells and NK-92, but lower levels in KHYG-1 EVs that translated into poor capacity for KHYG-1 EVs at targeting tumor cell lines. Further, we show that CD16-stimulated NK cells release low amounts of EVs devoid of cytolytic proteins. Importantly, EVs from cytokine-stimulated NK cells penetrate into the spheroid core, and tumor spheroid susceptibility to NK-cell derived EVs was linked to differential expression of the NKG2D ligands MICA/B, which was blocked with an anti-NKG2D antibody. We conclude that EVs from activated primary NK cells or NK-92 cells has the best potential to infiltrate and target solid tumors.

| S-EPMC9374793 | biostudies-literature

Dendritic cells Trigger IFN-γ secretion by NK cells independent of IL-12 and IL-18.

Project description:It is commonly believed that IL-12 produced by DCs in response to pathogens is the first signal that stimulates the production of IFN-γ by NK cells. However, IL-12 production by DCs in response to bacterial LPS depends on either engagement of CD40 by CD40L on activated T cells or IFN-γ from NK cells. This suggests that during the primary immune response, NK cells produce IFN-γ before IL-12 production by DCs. Here, using single-cell measurements, cell sorting and mouse lines deficient in IL-12, IL-23, type I IFN receptor and the IL-18 receptor, we show that a subset of BM-derived DCs characterized by low expression of MHC class II (MHCIIlow ) stimulates IFN-γ production by NK cells. The expression of Toll-like Receptor (TLR) 4 on DCs but not NK cells was required for such NK-derived IFN-γ. In addition, soluble factor(s) produced by LPS-activated MHCIIlow DCs were sufficient to induce IFN-γ production by NK cells independent of IL-12, IL-23, and IL-18. This response was enhanced in the presence of a low dose of IL-2. These results delineate a previously unknown pathway of DC-mediated IFN-γ production by NK cells, which is independent of commonly known cytokines.

| S-EPMC10608798 | biostudies-literature

IL-21-mediated reversal of NK cell exhaustion facilitates anti-tumour immunity in MHC class I-deficient tumours.

Project description:During cancer immunoediting, loss of major histocompatibility complex class I (MHC-I) in neoplasm contributes to the evasion of tumours from host immune system. Recent studies have demonstrated that most natural killer (NK) cells that are found in advanced cancers are defective, releasing the malignant MHC-I-deficient tumours from NK-cell-dependent immune control. Here, we show that a natural killer T (NKT)-cell-ligand-loaded tumour-antigen expressing antigen-presenting cell (APC)-based vaccine effectively eradicates these advanced tumours. During this process, we find that the co-expression of Tim-3 and PD-1 marks functionally exhausted NK cells in advanced tumours and that MHC-I downregulation in tumours is closely associated with the induction of NK-cell exhaustion in both tumour-bearing mice and cancer patients. Furthermore, the recovery of NK-cell function by IL-21 is critical for the anti-tumour effects of the vaccine against advanced tumours. These results reveal the process involved in the induction of NK-cell dysfunction in advanced cancers and provide a guidance for the development of strategies for cancer immunotherapy.

| S-EPMC5467212 | biostudies-literature

The IL-12- and IL-23-Dependent NK Cell Response Is Essential for Protective Immunity against Secondary <i>Toxoplasma gondii</i> Infection.

Project description:NK cells can develop cell-intrinsic memory-like characteristics. Whether they develop these characteristics during Toxoplasma gondii infection is unknown. We addressed this question and dissected the mechanisms involved in secondary NK cell responses using a vaccine-challenge mouse model of T. gondii infection. NK cells were required for control of and survival after secondary T. gondii infection. NK cells increased in number at the reinfection site and produced IFN-?. To test if these T. gondii experienced NK cells were intrinsically different from naive NK cells, we performed NK cell adoptive transfer into RAG2/c?-chain-/- mice, NK cell fate mapping, and RAG1-/- mice vaccine-challenge experiments. Although NK cells contributed to immunity after reinfection, they did not develop cell-intrinsic memory-like characteristics after T. gondii vaccination. The mechanisms required for generating these secondary NK cell responses were investigated. Secondary NK cell responses were CD4+ or CD8+ T cell independent. Although IL-12 alone is required for NK cell IFN-? production during primary T. gondii infection, in the absence of IL-12 using IL-12p35-/- mice or anti-IL-12p70, secondary NK cell responses were only partially reduced after reinfection. IL-23 depletion with anti-IL-23p19 in vivo also significantly reduced the secondary NK cell response. IL-12 and IL-23 blockade with anti-IL-12p40 treatment completely eliminated secondary NK cell responses. Importantly, blockade of IL-12, IL-23, or both significantly reduced control of parasite reinfection and increased parasite burden. Our results define a previously unknown protective role for NK cells during secondary T. gondii infection that is dependent on IL-12 and IL-23.

| S-EPMC6864276 | biostudies-literature

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