Membrane IL1α Inhibits the Development of Hepatocellular Carcinoma via Promoting T- and NK-cell Activation
Dandan LinLei LeiYonghao LiuYinsheng ZhangBo HuGuangming BaoYuan SongZiqi JinChunliang LiuYu MeiDedy SandikinYan WuLixiang ZhaoYu XiaoHaiyan Liu
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Abstract Hepatocellular carcinoma is a worldwide health problem with limited treatment options and poor prognosis. Inflammation associated with liver injury and hepatocyte regeneration can lead to fibrosis, cirrhosis, and eventually, hepatocellular carcinoma. IL1α is one of the most important inflammatory cytokines involved in inflammation and tumor development. IL1α presents as multiple forms in vivo, including precursor, propiece, membrane, and secreted forms, and their functions have been thought to be different. The role of membrane IL1α in hepatocellular carcinoma tumorigenesis is still not clear. Here, we examined the functions of membrane IL1α in murine hepatocellular carcinoma models. We found that membrane IL1α potently inhibited hepatocellular carcinoma tumor growth. Further studies showed that membrane IL1α promoted T- and natural killer (NK)–cell activation in vivo. IFNγ production by CD8+ T and NK cells was also increased as a result of membrane IL1α expression. Moreover, the cytotoxicity of the CTL and NK cells was also enhanced by membrane IL1α expression. Furthermore, in vitro studies demonstrated that membrane IL1α could directly activate T cells and NK cells in a cell contact–dependent manner. Conversely, depletion of both CD8+ T and NK cells suppressed the antitumor activity of membrane IL1α. Our studies demonstrated that membrane IL1α could promote antitumor immune responses through activation of T and NK cells. Thus, our findings provide new insights of IL1α functions during hepatocellular carcinoma development. Cancer Res; 76(11); 3179–88. ©2016 AACR.Keywords:
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Abstract NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3−), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3−) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-γ, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-γ, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-γ and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-γR knockout mice demonstrated that IFN-γ was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.
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Abstract Natural Killer (NK) cells are an important component of the innate immune system, capable of providing a fast and effective response against virally infected cells. NK cells are mainly characterized by their cytotoxic function and their ability to secrete cytokines. It has been shown that infant NK cells have decreased cytotoxicity and cytokine-secreting function, suggesting hyporesponsiveness of NK cells during the first year of life. Because NK cell activation is dependent on cytokine stimulation, we hypothesized that infant NK cells are hyporesponsive due to deficiencies in signaling following cytokine stimulation. We examined the activation of human infant and adult NK cells in response to IL2, IL12 or IFNα stimulation, cytokines important in NK survival and function. Using Phosflow analysis, we measured the phosphorylation of transcription factors, STATs, specific for the distinct cytokines. Cord blood NK cells showed significantly lower pSTAT activation when compared to adult NK cells when treated with IL2 or IFNα but not IL12. However, despite pSTAT4 activation in response to IL12, no nuclear translocation was observed by ImageStreamX Mark II analysis. NK cells in 6 and 12-month-old infants showed similar frequencies of pSTAT NK cells compared to adults when treated with IL2, IL12 or IFNα, suggesting that cytokine signaling in NK cells is age-dependent. Surprisingly, pSTAT activation was restored in cord blood NK cells when treated with a cocktail of IL2, IL12 and IL15. These results suggest that JAK/STAT signaling in infant NK cells is impaired at different steps in the pathway in response to specific cytokines.
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Natural killer(NK) cells,core components in human innate immunity,play a critical role in tumor cellular and antibody immunotherapy.NK cells can control tumor growth and metastasis through direct cellular cytotoxicity and secreted immune stimulatory cytokines.Multiple protocols currently aim to activate NK cells for utilization in cancer immunotherapy.Freshly isolated NK cells,transiently stimulated NK cells or lymphokine activated killer(LAK) cells are currently utilizated in autologous clinical trials.However,NK cells have displayed impaired functionality in cancer patients,which restricts their clinical effect.Several new protocols are available to improve clinical trial efficacy by increasing NK cells quantity and application of NK cells with high cytotoxicity.Large-scale production of clinical grade NK cells through long-term culture in vitro is one of the best ways in achieving these two goals.NK cells generated by certain methods with high quality and enough quantity have been translated into clinical use.The background of NK cells in immunotherapy,the status of NK cells in cancer patients,especially the new methods applied to expand cells in vitro are highlighted and discussed in this review.
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Summary We investigated the function of CD56 + CD8 + T cells (CD56 + T cells) and CD56 − CD57 + CD8 + T cells (CD57 + T cells; natural killer (NK)‐type T cells) and compared them with those of normal CD56 − CD57 − CD8 + T cells (CD8 + T cells) and CD56 + NK cells from healthy volunteers. After the stimulation with immobilized anti‐CD3 antibodies, both NK‐type T cells produced much larger amounts of interferon‐γ (IFN‐γ) than CD8 + T cells. Both NK‐type T cells also acquired a more potent cytotoxicity against NK‐sensitive K562 cells than CD8 + T cells while only CD56 + T cells showed a potent cytotoxicity against NK‐resistant Raji cells. After the stimulation with a combination of interleukin (IL)‐2, IL‐12 and IL‐15, the IFN‐γ amounts produced were NK cells ≥ CD56 + T cells ≥ CD57 + T cells > CD8 + T cells. The cytotoxicities against K562 cells were NK cells > CD56 + T cells ≥ CD57 + T cells > CD8 + T cells while cytotoxicities against Raji cells were CD56 + T cells > CD57 + T cells ≥ CD8 + T cells ≥ NK cells. However, the CD3‐stimulated proliferation of both NK‐type T cells was smaller than that of CD8 + T cells partly because NK‐type T cells were susceptible to apoptosis. In addition to NK cells, NK‐type T cells but not CD8 + T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3‐stimulated IFN‐γ production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57 + T cells in PBMC from donors. Our findings suggest that NK‐type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.
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In this unit, methodology is presented for measuring the capacity of human natural killer (NK) or lymphokine-activated killer (LAK) cells to lyse tumor cell targets. Cytotoxicity of these effector cells is evaluated in a short-term (51)Cr-release assay using NK-sensitive tumor cells as the targets for NK cells or NK-resistant tumor cells as the targets for LAK cells. In the basic protocol, a generic (51)Cr-release assay is described in which PBMC, purified NK cells or interleukin 2 (IL-2)-activated lymphocyte populations (LAK cells) are utilized as effector cells. Support protocols describe preparation of nonadherent tumor cells, cells obtained from malignant effusions, trypsinized tumor cells from adherent monolayer culture, or freshly isolated tumor cells from surgical specimens. All of these cell types can serve as the (51)Cr-labeled target cells in the basic protocol. The procedure for labeling target cells from any of these sources with (51)Cr is also provided.
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TAKAGI, S., MINAKUCHI, J., OKAWA, H. and YATA, J. Interferon-Induced Resistance of Tumor Target Cells against Lysis by Interleukin-2-Activated Killer Cells. Tohoku J. Exp. Med., 1989, 157 (2), 131-136-IFN-γ has been shown to decrease the susceptibility of target cells to NK cell-mediated cytotoxicity. In this report, the effect of IFN-γ on the sensitivity of target cells to killing by various human lymphocyte cytotoxic activities such as NK/K, IL-2-augmented NK/K cell activity, and IL-2-activated killer activity were studied. Although NK-sensitive K562 cells showed marked resistance to NK cell activity as previously reported, the resistance was overwhelmed by augmentation of NK activity with IL-2. IL-2-activated killer cell activity, which can lyse NK-resistant tumor cell lines upon culture in IL-2, showed decreased cytotoxicity against most of the IFN-γ-treated target cells tested. By contrast, no decrease of target cell sensitivity to K cells was observed, even though K cells were treated with IL-2. These findings suggest that as far as NK-resistant tumor cells are concerned, an IFN-dependent mechanism inhibits the Fc receptor-independent mediators of tumor surveillance, but not Fc receptor-dependent ones. This should be considered when planning adoptive immunotherapy of IL-2-activated killer cells for human malignancy.
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Author(s): Park, So-Hyun | Advisor(s): Jewett, Anahid | Abstract: Natural Killer (NK) cells have a crucial role in immune surveillance against a variety of infectious microorganisms and tumors. NK cells are known to mediate direct cytotoxicity as well as antibody dependent cellular cytotoxicity (ADCC) against a variety of tumor cells. Also, they are known to regulate the functions of other cells by producing key cytokines and chemokines. In the tumor microenvironment, cytotoxic function of NK cells is suppressed by a number of distinct effectors and their secreted factors. It has been shown that many cancer patients have decreased peripheral blood NK cell function, so NK cell-based immunotherapy has been used as a treatment in order to enhance NK cell function. However, limited availability of NK cells and ability to expand has restricted development of NK cell immunotherapy. Overcoming NK cell tolerance against tumors by developing new ways of activating endogenous NK cells that increase the expression of ligands for activating NK cell receptors or that render them more sensitive to NK cell mediated killing is crucial.In this study, we found the novel way to expand NK cells and the functionality of NK cells generated under this condition demonstrated enhanced expression of activating NK receptors. Also, significant cytotoxic killing potential after culture was discovered as well as augmented cytokine secretion. Therefore, these expanded NK cells are highly functional in comparison to primary NK cells. Through the help of cytokines, sAJ2 bacteria and osteoclast, NK cells can be expanded and activated in vitro and furthermore, these expanded NK cells can be used to target tumors in vivo. Expanded NK cells can be used in combination with other treatment modalities, potentially leading to synergistic antitumor activities.
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