The Absence or Overexpression of IL-15 Drastically Alters Breast Cancer Metastasis via Effects on NK Cells, CD4 T Cells, and Macrophages This information is current as of September 25, 2021. Amy Gillgrass, Navkiran Gill, Artem Babian and Ali A. Ashkar J Immunol published online 29 October 2014 http://www.jimmunol.org/content/early/2014/10/29/jimmun ol.1303175 Downloaded from Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 29, 2014, doi:10.4049/jimmunol.1303175 The Journal of Immunology The Absence or Overexpression of IL-15 Drastically Alters Breast Cancer Metastasis via Effects on NK Cells, CD4 T Cells, and Macrophages Amy Gillgrass, Navkiran Gill, Artem Babian, and Ali A. Ashkar IL-15 is a cytokine that can affect many immune cells, including NK cells and CD8 T cells. In several tumor models, IL-15 delays primary tumor formation and can prevent or reduce metastasis. In this study, we have employed a model of breast cancer metastasis to examine the mechanism by which IL-15 affects metastasis. When breast tumor cells were injected i.v. into IL-152/2, C57BL/6, IL- 15 transgenic (TG) and IL-15/IL-15Ra–treated C57BL/6 mice, there were high levels of metastasis in IL-152/2 mice and virtually no metastasis in IL-15 TG or IL-15–treated mice. In fact, IL-152/2 mice were 10 times more susceptible to metastasis, whereas IL- 15 TG mice were at least 10 times more resistant to metastasis when compared with control C57BL/6 mice. Depletion of NK cells Downloaded from from IL-15 TG mice revealed that these cells were important for protection from metastasis. When NK cells were depleted from control C57BL/6 mice, these mice did not form as many metastatic foci as IL-152/2 mice, suggesting that other cell types may be contributing to metastasis in the absence of IL-15. We then examined the role of CD4 T cells and macrophages. In IL-152/2 mice, in vivo depletion of CD4 T cells decreased metastasis. The lack of IL-15 in IL-152/2 mice, and possibly the Th2-polarized CD4 T cells, was found to promote the formation of M2 macrophages that are thought to contribute to metastasis formation. This study reveals that whereas IL-15 effects on NK cells are important, it also has effects on other immune cells that contribute to http://www.jimmunol.org/ metastasis. The Journal of Immunology, 2014, 193: 000–000. nterleukin-15 is a cytokine that has effects on cells of the promote tumor cell death due to their production of NO, proin- innate and adaptive immune system. It is most known for its flammatory cytokines, efficient Ag presentation, and phagocytic I effects in promoting the survival, differentiation, and acti- abilities (10, 11). In contrast, the alternatively activated group of vation of NK cells, as well as promoting memory CD8 T cell macrophages (M2), of which tumor-associated macrophages responses (1–3). Because both NK cells and CD8 T cells are (TAMs) are a member, has more of a role in tumor promotion (10, important in tumor defense and correlate with survival in various 12). It functions to promote angiogenesis, tumor cell invasion, and cancers, there has been extensive interest in IL-15 as an immu- intravasation, and it plays a role at the metastatic site (11). by guest on September 25, 2021 notherapy (4–6). The effects of IL-15 have been assessed in many Macrophages are very plastic and are able to travel between M1 tumor models, and it has been found to be effective against pri- and M2 states depending on the microenvironment in which they mary tumors as well as metastasis (7–9). In many cases, the are found (13). M1 macrophages are created in environments with mechanism of protection from metastasis was not evaluated. stimuli such as IFN-g and LPS (14). M2 is a group of macro- When investigated in models of metastasis, NK cells were most phages that can develop under the influence of IL-4/IL-13, im- frequently the cell type identified that was important for protection mune complexes, IL-10, TGF-b, or glucocorticoids (14). (7, 9). Although the role of macrophages at the primary tumor is well In recent years, it has become apparent that macrophages are established, a lot less attention has been paid to the role of mac- very important cell types involved in the promotion of metastasis. rophages at the metastatic site. Kaplan et al. (15) found that after Classically activated macrophages, also known as M1, are able to primary tumors have been established, but before metastasis has occurred, there are premetastatic sites in which there are clusters of vascular endothelial growth factor receptor+ hematopoietic Department of Pathology and Molecular Medicine, McMaster Immunology Research + Center, McMaster University, Hamilton, Ontario L8N 3Z5, Canada bone marrow progenitors. CD11b myeloid cells are also attracted Received for publication November 27, 2013. Accepted for publication October 5, to these premetastatic niches, and these cells are important for 2014. establishing metastasis (16). Recently, to investigate the role of This work was supported by grants from the Canadian Breast Cancer Foundation. macrophages at the metastatic site, several groups have used the A.G. was the recipient of Ph.D. fellowships from the Canadian Institutes for Health injection of tumor cells i.v. to mimic tumor metastasis. In models Research and the Canadian Breast Cancer Foundation; N.G. was awarded an Ontario Graduate Scholarship; and A.A.A. was the recipient of Canada’s Research-Based such as this, macrophages are required for efficient tumor cell Pharmaceutical Companies/Canadian Institutes for Health Research Career Award seeding as well as growth at the metastatic site (17, 18). in Health Research. Macrophage polarization is dependent upon the signals it The PCR array presented in this article has been submitted to the Gene Expression receives in its microenvironment. It is, of course, no wonder that Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61858) under ac- cession number GSE61858. CD4 T cells play a role in macrophage polarization. Th1 CD4 Address correspondence and reprint requests to Dr. Ali A. Ashkar, McMaster Uni- T cells produce IFN-g, which promotes M1 macrophage polari- versity, 1280 Main Street West, MDCL 4074, Hamilton, ON L8N 3Z5, Canada. zation, whereas Th2 or regulatory T cells produce IL-10, IL-4, or E-mail address: [email protected] IL-13, which promotes M2 macrophage polarization (19). In fact, Abbreviations used in this article: MT, middle T; TAM, tumor-associated macro- in mouse models that are Th2 biased, knockout of CD4 T cells phage; TG, transgenic. promotes the formation of macrophages with a more M1 pheno- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 type (20, 21). Macrophages also influence the polarization of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303175 2 THE EFFECTS OF IL-15 ON BREAST CANCER METASTASIS T cells themselves, with M2 macrophages promoting the forma- in PBS and resuspended as 0.5 3 106–1 3 106 per 400 ml sterile PBS. Cells 2/2 tion of T regulatory cells (22). Recently, it was discovered that, in with ,5% cell death were injected via the tail vein of IL-15 , C57BL/6, or a spontaneous model of breast tumor formation, CD4 T cells IL-15 TG mice. Mice were monitored for endpoint, and 13 d postinjection, mice were euthanized and lungs were harvested. Lungs were fixed in 2% expressing IL-4 were able to promote invasion and metastasis paraformaldehyde for 48 h. Sections were then stained with H&E for analysis due to their effects on promoting M2 phenotype in TAMs (20). In of lung tumor metastasis. The level of metastasis was assessed while blinded this case, loss of CD4 T cells actually decreased the amount of in ImageJ. In brief, in an entire lobe of lung, per each experimental animal, metastasis observed (20). Other studies have also indicated that a threshold algorithm was applied to assess tumor area (manually checked for accuracy). Total lung area (with lumen areas subtracted) was also assessed, CD4 T cells may play a role in promoting metastasis, although via and percentage of tumor area was calculated by the following formula: different mechanisms (23, 24). The effect of IL-15 on macrophages and CD4 T cells is an area Percent tumor area ¼ðtumor area=total lung areaÞ 3 100 that has not been studied thoroughly. It is known that IL-15 is Melanoma cells (B16F10) were also used and were prepared in the same produced from human monocytes upon LPS activation (25). manner as MT cells. A total of 1 3 105 cells was injected in the tail vein of Several in vitro studies on various sources of macrophages IL-152/2 and C57BL/6 control mice.