Effect of interleukins (IL-2, IL-15, IL-18) on receptors activation and cytotoxic activity of natural killer cells in breast cancer cell

Abstract Introduction: Breast cancer is one of the leading cause of cancer deaths in women. Metastasis in BC is caused by immunosurveillance deficiency, such NK cell maturation, low NK activity and decreasing cytotoxicity. This study was performed to improve activating receptors and cytotoxicity of NK cells using interleukins (ILs). Methods: Human recombinant IL-2, -15, and -18 were used to induce NK cells. We measured the activating and inhibiting receptors, proliferation activity of NK cells, and the cytotoxicity of NK cells on BC cells (MCF7). The effects of ILs were tested on the NK cell receptors CD314, CD158a and CD107a with flowcytometry, proliferation at various incubation times with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and concentrations of TNF-α and IFN-γ by NK cells with ELISA. Results: ILs increased NK cell receptor levels (CD314, CD158a, and CD107a) at 24 hours of incubation. ILs increased NK cell viability, which increased with longer incubation. Moreover, ILs-induced NK cells inhibited proliferation in MCF7 cells, as well as increased TNF-α, IFN-γ, PRF1 and GzmB secretion. Conclusion: IL-2, IL-15, and IL-18 improved activating receptors and proliferation of NK cells. IL-induced NK cells increased TNF-α, IFN-γ, PRF1 and GzmB secretion and cytotoxic activity on BC cells. High NK cell numbers increased BC cell growth inhibition.


Introduction
Breast cancer (BC) is the leading cause of cancer deaths in women 1 , accounting for approximately 43.3% 2 . Mor-tality of most patients with solid tumors is due to metastatic spread to other organs 1 . Metastasis occurs when tumor cells acquire invasive features 3 and the ability to escape from antitumor immunity 4,5 . Defects in antitumor immunity may also facilitate BC occurrence 6 . Metastasis in BC is caused by deficient immunosurveillance, including impairment of NK cell maturation, low NK cell counts in peripheral blood mononuclear cells (PB-MCs), significantly lower NK activity in patients with BC than in healthy individuals 7 , decreased cytotoxic function 8,9 , NK abnormalities 8 , poor tumor infiltrate 10,11 , low NK cell numbers in tumors due to their inefficient homing into malignant tissues 8 , defective expression of activating receptors such as NKG2D, NKG2C, NKp30, NKp46, CD161, CD56 dim , CD16, DNAM-1, and CD69 and high immunosuppression, namely overexpression, of inhibitory receptors CD158a, CD158b, and NK-G2A 8 . One cancer therapy is to stimulate NK cell function and combine them with other agents to boost anti-cancer activity 12 . NK cells as immunomodulators can be activated using interleukin into lymphokine-activated killer cells (LAK). NK cells respond to a variety of cytokines, such as IL-2, IL-12, IL-15, IL-18, IL-21, and Type I Interferons (IFNs), discretely or in combination with each other or with other modulators 13 , and tumor necrosis factor (TNF) 14 , which increases their cytolytic, secretory and anti-cancer functions 15 . Through its interaction with NK cells, IL-2 treatment was related with favourable result in various cancer type, making it the first effective immunotherapy for human cancer 16 .
Immunotherapy using NK cells can be used to obtain the large and sufficient numbers of functional NK cells necessary for clinical therapy. The number, purity and state of NK cell proliferation and activation are key factors in immunotherapy 17 . NK cells are known as necessary effectors in suppressing cancer proliferation 18 . Therefore, the focus of recent cancer therapy has been to promote and develop NK cells as drugs 9 , using NK effectors such as cytokines 8 .
This study was conducted to evaluate the effect of inducing interleukins (IL-2, IL-5, and IL-18) on NK cells toward the following: i) improvement of NK cell activating receptors, including CD314, CD158d and CD107a; ii) increased proliferation of NK cells; iii) NK cell secretion of factors such as IFN-γ and TNF-α; iv) secretion of IFN-γ, TNF-α, perforin (PRF1) and granzyme B (GzmB) co-cultured NK and BC cells inhibition of BC cell proliferation.
Detection of NK cell receptors using fluorescence activated cell sorting (FACS) NK92 cells with a density of 5x10 5 /well were cultured in complete medium containing RPMI 1640, 10% FBS, and 1% antibiotic/antimycotic incubated at 5% CO 2 , 37 °C for 24 h. The NK cells were induced with IL-2, IL-15, and IL-18 (10 ng/ml) and incubated for 24 h. The medium was collected, and 1x10 5 -1x10 6 NK cells were added to 500 ml of FACS Buffer (PBS + 2% FBS). Furthermore, NK receptors were stained with the surface markers CD314 (Ms anti-human, Invitrogen 521927), CD107a (Ms anti-human, Invitrogen 519297), and CD158d (Ms Anti-human, Invitrogen 523653) in single and multiple staining, followed by incubation in a dark room at 4 °C for 30 min. The cells were washed twice using FACS Buffer. NK receptors were stained with CD314, CD158d, or CD107a antibody conjugated with PE. The receptors were analyzed by flow cytometry with a MACSQuant® Analyzer. The experiments and measurement of surface markers were performed in triplicate 19,20 .

Effector molecules of ILs-induced NK cells
ILs-induced NK cells at a density of 1x10 5 cells/well were grown and maintained in NK medium and incubated at 5% CO 2 , 37 °C for 24 h. Cells and medium were centrifuged at 500 g for 4 min, and the culture supernatant was preserved at -80°C for subsequent assays, such as for IFN-γ and TNF-α 22,23 . IFN-γ secretion from NK cells was measured using a commercial enzyme-linked immunosorbent assay kit (Human ELISA KIT, Biolegend 430104) and the TNFα secretion from NK cells using a human ELISA Kit (Human ELISA KIT, Biolegend 430104) following the manufacturer's recommendation.

Growth inhibition of ILs-NK cells on breast cancer cells
This research method was adopted and modified based on the previous research by Parihar et al. (2002) and Lu et al. (2008) 22,23 . NK cells at a density of 1x10 6 / well were grown and maintained in NK medium, and cells were treated with 5 and 10 ng/ml of IL-2, IL-15, or IL-18 daily and incubated at 5% CO 2 , 37°C for 96 h, resulting in IL-induced NK cells. The BC cell line MCF7 (ATCC® HTB22™) was obtained from Aretha Medika Utama Biomolecular and Biomedical Research Center, Bandung, Indonesia, plated at a density of 1x10 4 /well and cultured on 96-well plates in 100 µl of complete medium (Dulbecco's Modified Eagle's Medium (DMEM high glucose; Gibco 11995065), 10% FBS, 1% antibiotic/antimycotic) and incubated at 5% CO 2 , 37°C for 24 h. The following day, culture supernatant was aspirated, and the cells were washed with PBS (Invitrogen 1740576) and replaced with complete medium (DMEM + 1% antibiotic/antimycotic, 10% FBS). Cells were incubated at 5% CO 2 , 37°C for 24 h. For the BC cells treated with NK cells in the number-dependent experiment, the ratios of BC cells to IL-induced NK cells were 1 : 5 (10,000 : 50,000), 1: 2 (10,000 : 20,000), 1 : 1 (10,000 : 10,000) and 2 : 1 (10,000 : 5,000). The medium comparison of cocultured cells was based on a preliminary study, and the best medium comparison of NK medium and BC medium was 1:1. The controls were the single cultures of NK92MI or MCF7. The co-cultured NK and BC cells were incubated at 5% CO 2 , 37°C for 24 h, 48 h, 76 h, or 96 h. To determine the growth inhibition or cytotoxic effects of NK cells toward BC cells using MTS assays, 20 µl of reagent cell titer 96 ® Aqueouse one solution assay (Promega G3580) was added to each well and incubated for 3 h. The cell absorbance was measured at 490 nm wavelength (Multiskan Go, Thermo Scientific. The inhibitory effect of IL-NK cells toward BC cell viability was calculated according to the following formula 23 :

Statistical analysis
Statistical analysis was conducted using SPSS software (version 16.0). Data were presented as Mean±Standard Deviation. Significant differences among treatments were determined using the one-way Analysis of variance (ANOVA) and p < 0.05 were considered as statistically significant, along with Tukey honestly significant difference post hoc test and 95% confidence interval.

Effect of ILs toward NK cell characteristics
To determine the effect of human recombinant ILs toward NK receptors, we evaluated the NK receptors including CD314, CD107a and CD158d (Figures 1). The data showed that ILs (IL-2, IL-15, and IL-18) significantly up-regulated CD314 and CD107 and that IL-15 and IL-18 significantly up-regulated CD158d, but IL-2 did not significantly up-regulate CD158d. African Health Sciences Vol 20 Issue 2, June, 2020 Effect of ILs toward NK cell proliferation NK cells require effectors to activate NK cells; thus, this study was conducted to evaluate the effect of ILs (IL-2, IL-15, and IL-18) toward NK cell numbers. We used ILs at levels of 5 and 10 ng/ml, and NK cell proliferation was calculated at various incubation times (24,48,72, and 96 h; Table 1). The data show ( Table 1) that ILs (IL-2, IL-15, and IL-18) increased NK cell proliferation and that proliferation was lower at 24 h of incubation compared to 48 h, 72 h, and 96 h of incubation.
A higher concentration (10 ng/ml) of ILs resulted in higher proliferation compared to 5 ng/ml.

Effect of ILs toward IFN-γ, TNF-α, PRF1 and GzmB in co-cultured breast cancer and NK cells
This study was aimed to measure the cytokines including IFN-γ, TNF-α, and cytolytic enzymes such as PRF1 and GzmB, which are secreted by co-cultured MCF7 and NK cells ( Table 2). The data in Table 2, show that MCF7 cells secreted low levels of PRF1 (20.00 pg/ml) and GzmB (13.33 pg/ml) and produced low levels of IFN-γ (1.83 pg/ml) and TNF-α (1.98 pg/ml). Co-cul-ture of MCF7 and non-activated NK cells increased IFN-γ, TNF-α, PRF1, and Gzm. Higher NK cell numbers in co-cultured cells increased the cytokines and cytolytic enzyme secretion of both non-activated and activated NK cells. IL-15 and IL-18 were the best effectors to induce NK cells to secrete IFN-γ, TNF-α, PRF1, and GzmB. The highest levels of IFN-γ, TNF-α, PRF1, and GzmB in co-cultured MCF7 and NK cells were secreted from co-cultured MCF7 and IL18-NK cells at a ratio of 1:5.

Effect of ILs toward BC proliferation in co-cultured BC and NK cells
To investigate NK cells as immunomodulators to kill and inhibit BC cell proliferation, we performed this research using co-cultured MCF7 and NK cells at various ratios. The effect of NK cells at various ratios can be seen in Table 3. The data in Table 3, showed that the ratio of BC and NK cells determined cancer cell proliferation, and the NK cell number affected cytotoxic potency on cancer cells. The growth inhibition among the MCF7 : NK ratio was significantly different. Higher NK cell numbers were more toxic than lower NK cell numbers, and the lowest NK cell number demonstrated the lowest growth inhibition on MCF7. The highest growth inhibition of NK cells was obtained with the highest NK numbers, with the MCF7: NK ratio of 1:5.
The IL inducers (IL-2, IL-15, and IL-18) did not exhibit significant differences in the inhibition of cell proliferation.
African Health Sciences Vol 20 Issue 2, June, 2020 with IL-12, IL-15, and IL-18. A brief (16-hour) pre-activation with IL-12, IL-15, and IL-18, followed by rest in vitro for 1-6 weeks, resulted in enhanced functionality, including IFN-γ production following re-stimulation with cytokines, or exposure to leukemia targets 28,29 . IL-2, IL-12, IL-15 and IL-18, applied systemically and for ex vivo activation and expansion of NK cells, have improved NK cell antitumor activity by increasing the expression of NK cell activating receptors and by inducing cytotoxic effector molecules 30 . IL-2 significantly increased KIR2DL1, KIR2DL2, and KIR3DL2 receptors on their surface by 30% vs. 22%, 37% vs. 30%, and 24% vs. 14%, respectively 31 . IL-12 alone or in combination with IL-18 significantly induced NK cell activity and CD107a degranulation marker expression in MM 32 . Resting NK cells typically express very low or undetectable levels of KIR2DL4 on their cell surfaces. Activation of NK cells with IL-2 and feeder cells resulted in a transient increase in the level of cell surface KIR2DL4 33 .
ILs increased NK proliferation, and a longer periodic incubation significantly increased NK viability (Table  1). These data were validated with previous research showing that IL15 efficiently triggered the activation and proliferation of NK and CD8+ T cells 34,35 . NK cell activity is regulated by cytokines such as IL-2, IL-12, IL-15, IL-18 and type I interferons (IFNs) 36,37 . NK cells treated with IL2 (IL2-NK) induced the proliferation of injected NK cells in the lung and increased the overall survival of mice with osteosarcoma lung metastasis 38 .
The NK cell expansion has been attempted using cytokines such as IL-2 and IL-15 39 , which are required for both NK cell maturation and survival 40 . IL-2 is the main cytokine that stimulates NK cell activation and proliferation 41 . NK cells also exhibit memory-like properties, and cytokine-induced memory-like (CIML) NK cells are generated via brief pre-activation with IL-12, IL-15, and IL-18 and later exhibited enhanced functionality upon re-stimulation 42 .
African Health Sciences Vol 20 Issue 2, June, 2020  Co-cultured BC cells (MCF7) and activated NK cells released higher levels of IFN-γ, TNF-α, PRF1, and GzmB compared to non-induced NK cells. Interleukins (IL-2, IL-15, and IL-18) increased the secretion of IFN-γ, TNF-α, PRF1, and GzmB; this result was in line with previous research that demonstrated an increase in the production of IFN-γ and TNF-α in CD107a of co-cultured NK-92 cells with cancer cells (HeLa, SiHa, and C-33A) pre-treated with the antitumor HO-1 inhibitor (SnPP). Antitumor agents increased IFN-γ and TNF-α in co-cultured NK cells and cancer cells 19. Expression of apoptosis-related proteins such as cleaved caspase-3 and Bax was increased in cancer cells co-cultured with NK-92 cells 20 . NK cells kill cancer cells by the release of cytoplasmic granules that contain a number of proteins, such as PRF1 and Gzm, which lyse target cells 20 . NK cells release the membrane-disrupting protein, PRF1, and proteolytic serine proteases, Gzm, from secretory granules 57 . The secretory pathways in NK cells ultimately regulate the separate lytic and regulatory capacity of these cells at the frontline of antitumor responses 58 . When K562 target cells and NK cells were co-cultured at ratios of 5:1 or 1:1, after 60 min, NK cells produced the cytokines IFN-γ and TNF-α 58 .
NK cells have anticancer potency, and higher NK cell numbers significantly increased cytotoxic activity (Table   3). These data were verified by previous studies showing that these cells are major effector cells of innate immunity and are generally thought to play a fundamental role in antitumor responses 8,59 . NK cells control tumor growth and metastasis diffusion in vivo 50 . There are low NK cell numbers in tumors due to their inefficient homing into malignant tissues 8 . Decreasing NK cell numbers are observed in PB of cancer patients; therefore, NK cells decrease in tumor infiltrate 8 . The activity and numbers of NK cells need to be enhanced for better efficacy 60 . NK cell infiltration in solid tumors was associated with a better prognosis 61 . NK cells need to be isolated and expanded in sufficient numbers for them to act as effector cells 60 . The comparison of effector and tumor determined NK cell cytotoxicity. A previous study showed that the lowest ratio (1.25:1) of effector and cervical epidermoid carcinoma (CaSki), effector and grade II, human cervix squamous cell carcinoma (SiHa) resulted in the highest viable cell target, while the highest ratio (20:1) resulted in the lowest viability or highest inhibition of target cells 62 . In mice lacking IL-12 and IL-18 cytokines, the cytolytic activity of the NK cells is further impaired, indicating that the cytolytic activity of NK cells is synergized by IL-12 and IL-18 in vivo 19 . The NK cells kill cancer cells through at least three mechanisms. NK cells can use the PRF/ Gzm-containing granule exocytosis pathway, the nitric oxide (NO) pathway and the death receptor-ligand pathway. The PRF1/Gzm pathway is the principle pathway by which NK cells kill cancer cells 63 .

Conclusion
IL-2, IL-15, and IL-18 improved activating receptors and proliferation of NK cells. IL-induced NK cells increased TNF-α, IFN-γ, PRF1, and GzmB secretion and cytotoxic activity on BC cells. High NK cell numbers increased BC cell growth inhibition.