Background

Natural Killer (NK) cells were discovered almost 40 years ago due to their ability to kill tumor cells with no prior sensitization [1]. Since then, extensive knowledge has been gained about their instrumental role in tumor immunosurveillance [2]. NK cells are capable of killing tumor cells via multiple mechanisms. The ability of an NK cell to kill another cell is controlled by a balance of activating and inhibitory receptors expressed on their cell surface that allow the NK cells to sense self versus damaged cells [3]. Recently, it has been shown that in addition to preventing tumor formation, NK cells can eradicate large solid tumors [4] and kill mammary cancer stem cells [5]. Unfortunately, in several malignancies, including breast cancer, NK cell activity as well as expression of activating receptors, is often suppressed [6,7]. Tumors promote this down regulation by the secretion of molecules such as TGF β and IL-10 [7-9]. Recent reports suggest that NK cells within the tumor may actually support tumor growth [10,11]. Alterations in NK cell activity are reversible, as NK cells rapidly respond to their environment [7,12]. The ability to shift the NK cell phenotype from inhibition/tumor promotion to activation will be essential for the use of NK cells against cancer.

IL-15 is a cytokine that has effects on both the innate and the adaptive immune system. IL-15 promotes the differentiation, proliferation and activation of NK cells and the formation of a subset of memory CD8 T cells [13-15]. This has been confirmed in IL-15 TG mice which have increased activated NK cells and increased proportions of memory CD8 T cells, whereas IL-15 KO mice lack NK cells and have decreased CD8 T cells [16,17]. The ability of IL-15 to promote both NK cell and CD8 T cell responses has led to interest in IL-15 as a cancer immunotherapy. Most in vivo studies investigating the effects of IL-15 have used subcutaneous engrafted or lung metastasis cancer models. For example, several studies found that IL-15 TG mice were resistant to engrafted tumor formation [18,19]. IL-15 has been administered by several routes and use of each of these methods has impaired tumor growth or metastasis [20-25]. The protection observed was either NK cell and/or CD8 T cell dependent [18-20,22]. While many treatment strategies have been successful in engrafted and metastatic models, it is unknown if this will translate into a spontaneous epithelial cancer model where tumors initiate and grow alongside an intact tolerized immune system.

In this study, we crossed IL-15 KO and IL-15 TG mice with a spontaneous breast cancer model (MT) to create IL-15 KO/MT and IL-15 TG/MT mice. MT mice express the polyoma MT antigen under the mouse mammary tumor virus long terminal repeat [26]. In MT mice, multifocal adenocarcinomas form and these frequently metastasize to the lung [26]. The MT model on a C57BL/6 background is a good model of human breast cancer as tumor formation is sequential and goes from focal hyperplasia to mammary intraepithelial neoplasms to carcinoma in situ and ends with multiple invasive tumors [27,1: Figure S2A). These NK1.1 + CD8 T cells were absent in IL-15 KO/MT tumors and much lower in MT tumors (Additional file 1: Figure S2A). To determine if these cells produced high levels of IFNγ, we isolated NK1.1+ cells from IL-15 TG/MT tumors and performed non-specific stimulation (CD3/28) in the presence of protein secretion inhibitors. 11-13% of CD3 + CD8+ NK1.1+ T cells produced IFNγ, a similar percent to that seen for total CD8 T cells that produced IFNγ (Additional file 1: Figure S2B, 5G). Therefore, in our model, these cells were capable of producing IFNγ, but were not the only CD8 T cells doing so.

Cells expressing NK1.1 are responsible for tumor destruction in IL-15 TG/MT mice

To determine which cell type(s) were responsible for anti-tumor responses in IL-15 TG/MT mice, we performed long term antibody depletion experiments with anti-NK1.1 or anti-CD8α antibody in IL-15 TG/MT mice. NK1.1 depletion efficiently depleted NK1.1+ cells within the spleen and tumor (Additional file 1: Figure S3A). IL-15 TG/MT mice that were depleted of NK1.1+ cells formed tumors faster and proceeded to endpoint more quickly than IL-15 TG/MT mice (p < 0.001, 0.0001, respectively) (Figure 6A). In fact, tumor formation closely followed what was observed in IL-15 KO/MT mice. In addition, histological analysis revealed that the tumor destruction in IL-15 TG/MT mice was absent when these NK1.1+ cells were absent (Figure 6C). The anti-CD8α antibody removed CD8 expressing cells from the spleen of IL-15 TG/MT mice, but only partially from the tumor (at least by 2/3 ie. 18% to 6%)(Additional file 1: Figure S3B). The majority of the CD3 + CD8+ T cells that were left in the depleted tumor were NK1.1+ (Additional file 1: Figure S3C). There were no statistically significant differences between the IL-15 TG/MT and the IL-15 TG/MT CD8 depleted mice (Figure 6B). Lastly, the tumors that formed in IL-15 TG/MT CD8 depleted mice had similar tumor destruction to that seen in normal IL-15 TG/MT mice (Figure 6C). Therefore, NK1.1+ cells play a major role in the tumor destruction and extension of survival in IL-15 TG/MT mice, whereas CD8 T cells, although activated phenotypically, play less of a role.

Figure 6
figure 6

The effect of NK1.1 and CD8α depletion on tumor formation. (A-B) IL-15 TG/MT mice were depleted with anti-NK1.1 (n = 6) (A) or anti-CD8α (n = 5) (B) antibody long term starting at 4 weeks of age. (A) In comparison to the tumor formation and survival curves for IL-15 TG/MT mice (n = 36 for percent tumor free, n = 28 for survival), NK1.1 depleted IL-15 TG/MT mouse tumors formed and progressed to endpoint more quickly. IL-15 TG/MT NK1.1 depleted mice were not different from IL-15 KO/MT tumor mice (n = 30). (B) In comparison to the tumor formation and survival curves for IL-15 TG/MT mice, CD8 depleted IL-15 TG/MT mouse tumors formed and progressed to endpoint at a similar rate. (C) Tumors that formed in IL-15 TG/MT NK1.1 depleted mice did not show the extensive destruction seen in normal IL-15 TG/MT tumors and in IL-15 TG/MT CD8 depleted mice. Arrows indicate areas of tumor destruction. **p < 0.01, ***p < 0.001.

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Adoptive transfer of CD8 T cells from IL-15 TG/MT mice does not lead to protection from tumor challenge

To further examine the impact of CD8 T cells from IL-15 TG/MT mice on tumor formation, we performed CD8 T cell adoptive transfers. C57BL/6 recipient mice were treated with cyclophosphamide to induce lymphopenia. 24 hours later CD8 T cells were isolated from the spleens/tumors of MT, IL-15 TG/MT and IL-15 TG mice, labelled with CFSE (5 × 106 spleen, 1 × 106 tumor) and injected IV. 24 hours after transfer, mice were challenged with a sub-cutaneous dose of fresh primary MT tumors from which immune cells had been removed. Mice were followed for tumor formation and endpoint. 8 days after challenge, several mice were sacrificed to ensure that the adoptive transfer was successful (4-8% of total CD8 T cells were CFSE positive - data not shown). There were no statistically significant differences in tumor formation or endpoint between control mice that received no CD8 T cells and mice that received CD8 T cells from MT, IL-15 TG/MT or IL-15 TG spleens or IL-15 TG/MT tumors (Additional file 1: Figure S4).

The effect of cytokines on the ability of human NK cells to kill a human breast cancer cell line

IL-15 overexpression in MT breast tumors created an environment where several cytokines that affect NK cell activation and proliferation were altered. We examined the expression of these cytokines in IL-15 TG/MT tumors versus MT or IL-15 KO/MT tumors and found that in addition to IL-15, both IL-12 and IL-18 were increased within IL-15 TG/MT tumors (Figure 7A/B). To determine if exposure to these 3 cytokines simultaneously would affect the ability of NK cells to kill breast tumor cells, we isolated NK cells from human PBMCs and stimulated them in an IL-15/IL-12/IL-18 rich environment for 16 hours. We then co-incubated them with a CFSE labelled MDA-231 human breast cancer cell line at various E:T ratios for 5 hours to determine their killing potential. The gating strategy used can be seen in Figure 7C and included exclusion of CD45+ NK cells, selection of CFSE+ tumor cells and finally the percent of CFSE+ tumor cells that were dead (7AAD+). MT cells alone were included as the level of basal MT cell death. In contrast to NK cells cultured in IL-2 alone, NK cells cultured in IL-15/IL-12/IL-18 were highly cytotoxic toward this breast cancer cell line and at E:T ratios of 10:1 reached specific lysis levels of 52% (Figure 7C/D).

Figure 7
figure 7

Cytokines in IL-15 TG/MT tumors and their effects on the ability of human NK cells to kill human breast cancer cells. (A & B) IL-12 and IL-18 are increased in IL-15 TG/MT tumors. (A) IL-12 ELISAs were performed on tumor homogenates from 6 size matched tumors per group. (B) 3 tumors from different mice in each tumor group were pooled (size matched between groups) and assayed for IL-18 cytokine levels via multianalyte protein analysis. (C & D) NK cells isolated from human PBMCs were cultured in IL-2 or IL-15/IL-12/IL-18 for 16 hours before being incubated with CFSE labelled MDA-231 cells for 5 hours in a killing assay. In the demonstration of the gating strategy in C) CD45+ NK cells were first removed from the analysis and then CFSE+ MT cells were selected. 7AAD was used to determine the percentage of the CFSE+ MT cells that were dead. Results at an E:T ratio of 1:10 are displayed (E:T = NKcell:MDA-231). MT alone is included to show spontaneous death levels which were used in the calculation of D. (D) Percent specific lysis of MDA-231 in the killing assay at various E:T ratios. Representative of 2 experiments. *p < 0.05.

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Discussion

While IL-15 has been under investigation as a cancer immunotherapeutic for the last decade, investigation has focused on tumor models that do not closely mimic spontaneous tumor formation in humans. It is known that injecting tumor cell lines subcutaneously or intravenously is a useful, but rather artificial system in which it is easier to develop immune responses to the tumor. In addition, there is a lack of studies that have examined the impact of IL-15 on solid epithelial tumors such as breast cancer. To examine the role of IL-15 in a more relevant model we utilized an immunologically tolerant mouse model of spontaneous mammary tumor formation (MT) and examined tumor formation in the absence of IL-15 (IL-15 KO/MT) or with IL-15 overexpression (IL-15 TG/MT). Overall, IL-15 TG/MT mice had increased survival when compared to either MT or IL-15 KO/MT mice. In contrast IL-15 KO/MT mice had faster tumor formation and decreased survival when compared to MT or IL-15 TG/MT mice. These results are similar to the anti-tumor effects of IL-15 that have been observed in other engrafted and metastatic models of melanoma and colon cancer, but it is one of the first reports of this in a spontaneous model of breast cancer [18,19,21].

IL-15TG/MT mice formed tumors, but these tumors had a very different phenotype than IL-15 KO/MT or MT tumors. This included large areas of cell death, a higher proportion of NK cells as well as increased CD8 T cell infiltration. Increased CD8 T cells or NK cells within the tumor is a positive prognostic factor in many human and mouse tumor types [32,36,37]. In many cases though, NK cells within the tumor express inhibitory receptors instead of activating receptors and have low cytotoxicity [7,38]. To examine NK cell phenotype, we identified NK cells using flow cytometry as NK1.1 + CD3- cells. NK1.1 is commonly used as a marker for NK cells in C57BL/6 mice. It is a member of the NKRP1 receptor family and while it is thought to be an activating receptor its’ ligand is unknown [39-41]. The NK cells within IL-15 TG/MT tumors possessed higher levels of both activating receptors (NKG2D, NKp46) and other markers of activation (CD69). Recently, ligands for NKG2D and NKp46 were found to be expressed on human primary breast tumors and breast tumor cell lines [9]. In addition, blockade of either NKp46 or NKG2D decreased the ability of NK cells to kill breast tumor cells that expressed ligands to these receptors [9]. We also found that there was a higher percentage of CD27high NK cells in IL-15 TG/MT tumors than in MT tumors. CD27 expression, in addition to CD11b expression, has been used to define mature mouse NK cells into subsets [30,42]. In progression from less mature to more mature: CD27lowCD11blow to CD27highCD11blow to CD27highCD11bhigh to CD27lowCD11bhigh [42]. Importantly, CD27high NK cells were found to have a higher degree of effector function, including cytotoxicity and cytokine production [30]. More recently it was found that CD27 on human NK cells could also be used to define NK cell subsets [43,44]. In contrast to what has been found with mouse NK cells, human CD27+ NK cells have been associated with low cytotoxic activity and high ability to secrete cytokines [43,44]. Overall, NK cells found within IL-15 TG/MT tumors are likely more capable of killing breast tumor cells than those found in MT tumors.

We observed increased CD8 T cells within IL-15 TG/MT tumors, but CD8 T cells within the tumor are not always functional as they can be anergic and/or exhausted [33,35]. While IL-15 KO/MT tumor CD8 T cells had high levels of exhaustion markers (PD-1) and lacked IFNγ production, IL-15 TG/MT CD8 T cells had very low levels of PD-1 and produced large amounts of IFNγ. This is in contrast to another report that found that treatment with IL-15 in a metastatic model of colon carcinoma led to increased PD-1 expression on CD8 T cells in the spleen [45]. It is likely that the short term administration of IL-15 or the model system used accounted for the discrepancies in our observations. In addition, a higher proportion of CD8 T cells in the IL-15 TG/MT tumors were CD44+ and CD62Lhigh, which are markers of central memory CD8 T cells. Central memory CD8 T cells are thought to be extremely effective in anti-tumor defence [31,46]. We also observed a high proportion of unique NK1.1+ CD8 T cells in IL-15 TG/MT tumors. This cell type has been previously identified as highly cytotoxic (high perforin/granzyme level) and able to produce large amounts of IFNγ [47,48]. In our model, while some of these cells produce IFNγ, they were not the major source. While we have not examined this here, it is interesting to speculate about whether these unique CD8 T cells developed in the tumor or migrated to the tumor from elsewhere. We do know that a low percentage of these cells can be found in IL-15 TG mice in other organs such as the spleen (data not shown), so they are not completely unique to the tumor environment. The ability of IL-15 to induce expression of other NK cell markers such as CD56 in human CD8 T cells has also been reported in a variety of models [49,50]. Thus, this effect does not appear to be limited to our mouse models or to one type of NK cell receptor.

In previous studies involving IL-15, protective effects were found to be NK cell or CD8 T cell dependent [18,19,22]. In IL-15 TG/MT mice, NK1.1 positive (includes NK cells, NKT cells, NK1.1+ CD8 T cells) but not CD8 positive cells were the most important cells for increased survival and tumor destruction. The CD8 depletion was substantial but not complete in the tumor and the majority of CD8 T cells that remained in the tumor were NK1.1+, indicating that this cell type was resistant to depletion via this method. It has previously been reported that these cells may be resistant to activation-induced cell death and this may contribute to the inefficient depletion [48]. Since CD3 + CD8 + NK1.1+ cells were removed by the NK1.1 depletion, we cannot rule out a role for this cell type in the tumor destruction of IL-15 TG/MT mice. The lack of contribution of CD8 T cells to increased survival was surprising due to the fact that they existed in such high numbers and were of the correct phenotype to fight cancer. To confirm this data, we performed a CD8 T cell adoptive transfer experiment. Despite the fact that CFSE labelled CD8 T cells were present in the spleen and tumor at endpoint (data not shown), there was no impact on survival from transfer of either IL-15 TG/MT, MT or IL-15 TG splenic CD8 T cells or IL-15 TG/MT tumor CD8 T cells after a subcutaneous primary MT tumor injection. It is possible that either this aggressive tumor formed too fast for the transferred CD8 T cells to have an impact or that the tumor rapidly lost MHC I expression to compensate for the presence of tumor specific CD8 T cells. Also, MT tumor formation has been found to be slightly different each time and different tumors may express different tumor antigens, some even lose expression of MT itself [51]. Thus, it is possible that despite taking MT tumor cells to inject from multiple mice and CD8 T cells from multiple mice and pooling them, they may still not have been specific for that tumor. In terms of MHC I loss, a similar phenomenon may be occurring in the spontaneous model. It is also possible that IL-15 overexpression may induce non-specific proliferation of CD8 T cells, not tumor specific responses [52]. This data indicates that overexpression of IL-15 can generate an anti-tumoral NK cell response that is effective at extending survival in the MT model.

Another promising finding revealed in this model was that overexpression of IL-15 appears to delay the formation of lung metastases. This observation, in a spontaneous model of breast tumor metastasis, strongly indicates that IL-15 has potential therapeutically to prevent metastasis. Previously, this has only been examined in injected models of metastasis. This may be very useful in a clinical setting in which metastasis is frequent and leads to significant increases in mortality.

Conclusions

IL-15 is a promising new cancer therapeutic that is well tolerated in primate models [53]. It appears to be superior to IL-2 as it has lower toxicity, does not increase T regulatory cells and induces higher levels of NK cell and CD8 T cell effector responses [53,54]. Based on the success of IL-15 in animal models, the first clinical trials have begun (NCT01727076, NCT01021059, NCT01572593) in multiple tumor types including melanoma, renal cell carcinoma and non-small cell lung carcinoma patients. Recently, there has been renewed interest in NK cells as a target to activate in the fight against breast cancer. It has been shown that human breast cancer cells express activating ligands as well as death-inducing receptors- both of which NK cells use to correctly identify their target cells [9,55]. In fact, expression of NKG2D ligands in human breast cancer was associated with a significant beneficial outcome [56]. It has also been established that NK cells are capable of eradicating a solid epithelial cancer (fibrosarcoma) and that they may also be able to target breast cancer stem-cell like cells [4,5]. Here, we found that when human NK cells were exposed to a similar cytokine environment to that found in IL-15 overexpressed MT tumors, they were highly capable of killing a triple negative breast cancer cell line. Other studies have reported that NK cells grown in IL-15, IL-12 and IL-18 are thought to display long term effector functions and may be memory-like NK cells [57,58]. These studies, along with our data, lends credence to the idea that stimulating innate immune cells such as NK cells can be effective clinically against breast cancer primary tumors and metastasis.