The Absence or Overexpression of IL-15 Drastically Alters Breast Cancer Metastasis via Effects on NK Cells, CD4 T Cells, and 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 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 , 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 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 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. polarization is dependent upon the signals it The PCR array presented in this article has been submitted to the 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 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 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. Lungs were harvested 14 d postin- (mouse peritoneal macrophages, RAW cells, human monocytes) jection and fixed in paraformaldehyde, and the number of visible nodules indicate that IL-15 is able to stimulate proinflammatory cytokine was assessed with a stereoscope. release (TNF-a, IL-1, IL-6, IL-8, MCP-1) as well as NO pro- IL-15/IL-15Ra treatment duction (26, 27). Others have also determined that IL-15 treat- A dose of IL-15 (500 mg; PeproTech) and IL-15Ra (1 mg; PeproTech) in ment promotes the formation of macrophages with higher 200 ml PBS per mouse was incubated for 30 min at 37˚C prior to injection costimulatory molecules (CD80/86) as well as increased MHCII i.p. into mice. C57BL/6 mice were treated with this dose of IL-15/IL-15Ra Downloaded from (28). The reported effects of IL-15 on CD4 T cells are rather 4 and 3 d prior to tumor cell injection, on the day of tumor cell injection contradictory. Early reports indicated IL-15 could prime CD4 and every 3–4 d till the end of the experiment. This dosing schedule had been previously tested in our laboratory (data not shown). A total of 1 3 T cells for Th1, although later it was found that it could induce 106 MT cells was injected i.v. Mice were monitored for endpoint, and 13 d Th1 or Th2 depending on other cytokines present (IL-12/IL-4, postinjection, mice were euthanized and lungs were harvested. Lungs were respectively) (29, 30). In addition, some report that it can prime fixed in 2% paraformaldehyde for 48 h. Sections were then stained with

naive T cells, whereas others report effects only on memory CD4 H&E for analysis of lung tumor metastasis. http://www.jimmunol.org/ T cells (29–31). Some find that IL-15 can promote the formation NO assay of T regulatory cells, but that it also increases the proliferation and cytokine secretion of IFN-g–producing CD4 and CD8 Under anesthetic, the peritoneal cavity of the mice was washed with RPMI 1640 (10% FBS, 1% penicillin/streptomycin, 1% HEPES, and 1% L-glu- T cells so that the T regulatory cells were unable to inhibit the tamine). Resultant cells were plated for 2–4 h, and only adherent cells were T cells (32, 33). In contrast, it was recently found that, in mice collected to be used in subsequent assays. A total of 1 3 105 or 2 3 105 that lack the receptor for IL-15 signaling (IL-15Ra2/2), CD4 peritoneal macrophages was plated in 96-well plates with RPMI 1640 T cells that were primed in this environment produced high (10% FBS, 1% penicillin/streptomycin, 1% HEPES, and 1% L-glutamine) or media supplemented with LPS (100 ng/ml; Sigma-Aldrich). Assay was levels of IL-10 (34). performed in triplicate. At 24 and 48 h, media was collected and combined In view of the recent literature, we wanted to determine the effect with Greiss reagent (0.04 g/ml; Sigma-Aldrich). Absorbance was measured by guest on September 25, 2021 of IL-15 on metastasis and the mechanism by which it promotes at 450 nm and compared with a standard curve of sodium nitrate. this effect. This will include whether IL-15 or the lack of IL-15, directly or indirectly, affects the polarization of macrophages and CD4 T cells and whether this would affect metastasis. We have employed a model of metastasis in which we inject breast tumor cells i.v. in mice that lack IL-15 (IL-152/2) and mice that over- express IL-15 (IL-15 transgenic [TG]). IL-152/2 mice lack NK cells, whereas IL-15 TG mice have increased NK cells (23 in C57BL/6; data not shown) (35, 36). This model of metastasis is frequently used to examine the later stages of metastasis when tumor cells have already entered the bloodstream. In this model, we will examine the roles played by IL-15, NK cells, CD4 T cells, and macrophages in lung metastasis.

Materials and Methods Animal models IL-152/2 and IL-15 TG mice were bred and maintained in the McMaster Central Animal Facility in clean rooms with a 12-h day/night schedule and standard temperature controls. Control C57BL/6 mice were ordered from Charles River. Procedures performed in this study were approved by the McMaster Animal Research Ethics Board and comply with the guidelines set out by the Canadian Council on Animal Care. C57BL/6 (Charles River), IL-152/2 mice (Taconic, Germantown, NY), and IL-15 TG mice (provided by M. Caligiuri, Ohio State University, School of Medicine, Columbus, OH) on a C57BL/6 background were used. FIGURE 1. IL-152/2 mice have increased lung metastasis. IL-152/2, Metastasis model C57BL/6, and IL-15 TG mice were injected with 5 3 105 MT cells i.v.. A cell line was established from a mouse mammary tumor virus/polyoma After 13 d, lungs were harvested, fixed, sectioned, and stained for H&E middle T (MT) C57BL/6 spontaneous breast tumor (Mayo Clinic, Scotts- (original magnification 3 1.6) (A). Percentage of lung area occupied by dale, AZ) grown in RPMI 1640 (10% FBS, 1% penicillin/streptomycin, 1% metastasis is reported in (B)(n = 5/group). Representative of three HEPES, and 1% L-glutamine). After brief trypsinization, cells were washed experiments. ***p , 0.001. The Journal of Immunology 3

48 h, the supernatant was collected and analyzed for cytokines. Control wells were included. Purity of CD4 T cells after enrichment was .85% via flow cytometry. ELISA IL-10 (DY417) and IL-13 (DY413) ELISAs were performed using Quantikine Murine Duoset Kits from R&D Systems (Minneapolis, MN). ELISAs were performed as per manufacturer’s instruction. Isolation of cells and RNA from lungs IL-152/2 mice or C57BL/6 mice were injected with 5 3 105 MT tumor cells i.v. Two days postinjection, lungs were perfused and harvested, and one lobe was used for RNA isolation and the other for cell isolation. For cell isolation, lungs were minced and digested in 150 U/ml collagenase I (Life Technologies) in HBSS for 1 h at 37˚C with shaking. Cells were then filtered and stained for CD16/32 (eBioscience) (1 in 100, 15 min, 4˚C), followed by staining for CD45, F480, CD11b, CD11c, GR1 or CD45, CD4, CD3 (BD Biosciences or eBioscience). CD45+ leukocytes were gated on first to begin analysis. To extract RNA, TRIzol (Invitrogen) was used initially (as per manufacturer’s instruction), followed by Qiagen RNeasy Mini Kit and on-column DNase treatment (RNase-Free DNase Set

79254; Qiagen). Subsequently, 1 mg RNA per sample was used to create Downloaded from cDNA following the procedure outlined in RT2 First Strand Kit 3304011; Qiagen). The cDNA was then used in a RT2 Profiler PCR Array for Mouse Cytokines and , as per manufacturers’ instruction (RT2 SYBR Green ROX PCR Mastermix; SABiosciences/Qiagen). This 96-well plate includes housekeeping , genomic DNA controls, reverse- transcription controls, and positive controls. The plate was run on the Applied Biosystems 7900 RT machine. Results were analyzed using the SABiosciences analysis tool, and all samples used passed the quality http://www.jimmunol.org/

2 2 controls inherent in these assays. Results are reported as fold up- or FIGURE 2. IL-15 / mice are more susceptible than control mice to downregulation, with two samples per group included in the analysis both MT and B16F10 cell metastasis, whereas IL-15 TG mice are more (accession GSE61858; link to data file: http://www.ncbi.nlm.nih.gov/geo/ resistant to MT metastasis. IL-152/2, C57BL/6 (A), and IL-15 TG (B) query/acc.cgi?acc=GSE61858). mice were injected with 1 3 104 to 1 3 106 MT cells i.v. After 13 d, lungs Statistical analysis were harvested, fixed, sectioned, and stained for H&E. Original magnifi- 2 2 cation 31.6. (C) IL-15 / and control C57BL/6 mice were injected with GraphPad Prisms was used for statistics. T tests or one-way ANOVAs were 1 3 105 B16F10 cells i.v., and 14 d later, lungs were harvested and performed, depending on the number of groups to be compared. For evaluated for metastatic tumor nodule formation (n = 5/group). *p , 0.05. ANOVAs, Bonferonni’s posttest was employed to compare groups. by guest on September 25, 2021

Ab depletions Results IL-152/2 mice have increased lung metastasis, whereas IL-15 Mice were given two injections, 1 day apart of either anti-CD4 (GK1.5; American Type Culture Collection) (100 mg) or anti-NK1.1 (PK136 TG mice and IL-15–treated C57BL/6 mice have decreased lung mouse IgG2, hybridoma HB191) (200 mg) Ab i.p. On day 4 after the first metastasis in comparison with control injection, MT cells were injected in the tail vein. CD4 depletion was IL-15 can impact, both directly and indirectly, many cell types that carried out once per week, and NK1.1 depletion was every 3–4 d to maintain the depletion for the course of the experiment. Efficiency of are thought to be important in the establishment of metastasis. To depletion was checked via flow cytometry. from each group were determine whether the absence or overexpression of IL-15 is able to analyzed for CD4+CD3+ T cells or NK1.1+ cells. affect the formation of metastasis, we used a mouse model mim- 2/2 Nonspecific CD4 stimulation icking breast tumor metastasis. IL-15 , C57BL/6, and IL-15 TG mice were injected i.v. with 5 3 105 MT cells (cell line established 2/2 Single-cell suspensions were created from spleens of IL-15 , C57BL/6, from a mouse mammary tumor virus-polyoma MT mouse breast and IL-15 TG mice. CD4 T cells were isolated using CD4 T cell enrich- 2/2 ment kit (Stem Cell) and plated at 5 3 105 in triplicate in 96-well plates tumor). The IL-15 mice had lungs with extensive metastasis that had been coated in purified CD3 (1 mg/ml)/CD28 (5 mg/ml) (eBio- (Fig. 1). At the same time point, there was greatly reduced meta- science) Ab overnight at 4˚C (washed in 23 PBS before plating). After static tumor burden in control mice, and metastasis was virtually

FIGURE 3. Treatment with exog- enous IL-15/IL-15Ra makes C57BL/ 6 mice resistant to metastasis. C57BL/6 (n = 7), IL-15/IL-15Ra– treated C57BL/6 (n = 7), and IL-15 TG (n = 2) mice were injected with 1 3 106 MT cells i.v. After 13 d, lungs were harvested, fixed, sec- tioned, and stained for H&E. Each picture is representative of a different mouse in that group (numbered 1–5). 4 THE EFFECTS OF IL-15 ON BREAST CANCER METASTASIS absent in IL-15 TG mice (Fig. 1). The difference between IL-152/2 Resistance to metastasis in IL-15 TG mice is NK cell dependent and C57BL/6 or IL-15 TG mice was statistically significant. To 2/2 IL-15 has very important effects on cells of both the innate and the explore the susceptibility of IL-15 mice, we reduced the amount adaptive immune system. It is known that IL-152/2 mice lack NK 3 4 of tumor cells injected down to 1 10 (Fig. 2A). Tumors began to cells, whereas IL-15 TG mice have increased NK cells and CD8 2/2 3 4 form in IL-15 mice at as low a dose as 5 10 ,whereasthey T cells (35, 36, 39). NK cells and CD8 T cells function in tumor 3 5 did not form in C57BL/6 mice until a dose of 5 10 . To delineate immunosurveillance and are able to kill tumor cells (39). To de- the differences between control and IL-15 TG mice, we increased termine whether NK cells or CD8 T cells are important to the 3 6 the amount of tumor cells injected (1 10 ) (Fig. 2B). In this resistance observed in IL-15 TG mice, we depleted IL-15 TG mice scenario, metastasis in the control mice was much higher and was with anti-NK1.1 or anti-CD8a Abs prior to and throughout the still absent in IL-15 TG mice (Fig. 2B). Lastly, to confirm the high 2/2 experiment (Fig. 4). Depletions with these Abs were effective degree of susceptibility to MT metastasis observed in the IL-15 (data not shown). A total of 5 3 105 MT tumor cells was injected 3 5 mice in an alternate tumor model, we injected 1 10 B16F10 i.v. The depletion of NK1.1+ cells increased metastasis to levels melanoma cells IV (Fig. 2C). Once again, a significantly higher degree of lung tumor formation was observed in the IL-152/2 mice in comparison with control C57BL/6 mice. We also examined what would occur in our model with short- term treatment of control C57BL/6 mice with exogenous IL-15/ IL-15Ra prior to and during the metastasis experiment. The combination of IL-15 with IL-15Ra was used for treatment, as it Downloaded from has been demonstrated to have higher activity, increased stabili- zation, and longer t1/2 than IL-15 alone (37, 38). We found that, at a dose of 1 3 106 MT cells i.v., there was extensive lung me- tastasis in all C57BL/6 mice, but virtually no metastasis in IL-15/ IL-15Ra–treated mice or IL-15 TG mice (Fig. 3). Thus, short-

term administration of IL-15 was able to protect from metastasis http://www.jimmunol.org/ formation. by guest on September 25, 2021

FIGURE 5. NK cells decrease metastasis in C57BL/6 mice and con- tribute to type 1 cytokines in the lung environment, but do not recapitulate the IL-152/2 phenotype. (A) C57BL/6 and IL-152/2 mice were depleted of NK cells (anti-NK1.1 Ab) or treated with PBS prior to injection with FIGURE 4. Depletion of NK cells in IL-15 TG mice promotes metas- 5 3 105 MT cells i.v. Depletion was continued throughout the experiment. tasis to a similar level as in IL-152/2 mice. (A and B) IL-15 TG mice were After 13 d, lungs were harvested and fixed for H&E sectioning (n =5/ depleted of NK cells or CD8 T cells prior to injection with 5 3 105 group). Original magnification 31.6. (B) Percentage of lung area occupied MT cells. Depletion of CD8 T cells had no effect on metastasis, whereas by metastasis is reported. (C) Lungs were isolated from IL-152/2 or depletion of NK cells promoted tumor formation (representative pictures C57BL/6 mice 2 d posttumor cell injection (5 3 105). RNA was extracted, shown, n = 5/depleted group, n = 4 IL-152/2, n = 3 IL-15 TG). H&E, cDNA was synthesized, and a PCR array was performed for cytokines and original magnification 3 1.6. (B) Percentage of lung area occupied by chemokines (n = 2/group). Fold downregulation in IL-152/2 versus metastasis is reported. *p , 0.05. C57BL/6 is reported. The Journal of Immunology 5 similar to that seen in IL-152/2 mice, whereas depletion of CD8 T cell phenotype, but conclusions regarding this have been mixed. T cells had no statistically significant effect on metastasis (Fig. 4). To determine whether CD4 T cells impacted metastasis in vivo, Therefore, NK cells are key mediators of the protection seen in IL- IL-152/2 mice were depleted of CD4 T cells before being injected 15 TG mice. i.v. with 5 3 105 MT tumor cells (Fig. 6). Depletion of CD4 T cells from IL-152/2 mice decreased tumor formation greatly in NK depletion in control mice increases metastasis, but not to 2/2 2 2 comparison with IL-15 mice (Fig. 6A, 6B). Therefore, CD4 the same level observed in IL-15 / mice T cells in IL-152/2 mice promoted tumor formation. It was also 2/2 To determine whether the lack of NK cells in IL-15 mice was found that CD4 T cells were a higher proportion of CD45+ leu- the factor that promoted increased metastasis in these mice, kocytes in the lung of IL-152/2 mice 2 d posttumor cell injection control mice were depleted of NK cells before being injected with in comparison with control lungs (Fig. 6C). Thus, they were 5 5 3 10 MT cells. Depletions were maintained for the duration of a highly prevalent immune cell present in the IL-152/2 mouse the experiment. We found that, although depletion of NK cells in lung microenvironment. control mice promoted metastasis, the trend in two separate 2/2 experiments was that there was still less metastasis than was ob- IL-15 mice have M2-polarized macrophages, whereas served in IL-152/2 mice (Fig. 5A, 5B). Thus, it seemed likely that IL-15 TG mice have M1-polarized macrophages there were other factors involved that promoted metastasis in IL- TAMs or M2 macrophages are an important cell type for promotion 152/2 mice. We also assessed the differences in cytokine ex- of metastasis (17, 42). Many studies have shown that, at the pri- pression in the lung environment of IL-152/2 lung versus control mary site, TAMs promote both invasion and metastasis and are

C57BL/6 lung 2 d posttumor cell injection (accession GSE61858; correlated with poor prognosis (11). It has been previously found Downloaded from link to data file: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? in the same model of metastasis used in this work that genetic acc=GSE61858). The main difference between these lungs in- or chemical ablation of macrophages decreases both metastatic volved a decrease in CCL5 (RANTES; 4548), , IFN-g, seeding and growth (17). To determine whether IL-15 affects the and XCL1 (lymphotactin) in IL-152/2 mice (Fig. 5C). CCL5, polarization of macrophages, we analyzed the ability of peritoneal IFN-g, and XCL1 have all been identified as being expressed macrophages from IL-152/2, control, and IL-15 TG mice to

when NK cells are activated in response to infections such as produce NO in response to LPS (Fig. 7A). IL-15 TG macrophages http://www.jimmunol.org/ Listeria or murine CMV (40, 41). produced the highest amounts of NO, which indicates a M1 2/2 2/2 phenotype, whereas IL-15 mice produced the lowest amount CD4 T cells in IL-15 mice promote metastasis of NO. We also examined the myeloid population present in the Recently, several reports have indicated that CD4 T cells play a role lungs of IL-152/2 and C57BL/6 mice at 2 d posttumor cell in- in promoting metastasis (20, 23, 24). IL-15 is known to affect CD4 jection (Fig. 7B). Cells in the lung were stained for F4/80 to by guest on September 25, 2021

FIGURE 6. In IL-152/2 mice, CD4 T cells promote metastasis. IL-152/2 mice were depleted of CD4 T cells (anti-CD4a Ab) or treated with PBS prior to injection with 5 3 105 MT cells i.v. Depletion was continued throughout the experiment. After 13 d, lungs were harvested, fixed, sectioned, and stained for H&E. Original magnification 31.6. (A) Percentage of lung area occupied by metastasis is reported in (B)(n =5/ group). (C) Lungs were isolated from IL-152/2 or C57BL/6 mice 2 d posttumor cell injection (5 3 105). Lungs were digested, and the resultant cells were an- alyzed via flow cytometry for CD45, CD3, and CD4. Results are reported as percentage of CD45+ cells in the lung. *p , 0.05. 6 THE EFFECTS OF IL-15 ON BREAST CANCER METASTASIS Downloaded from

FIGURE 7. IL-15 affects macrophage polarization and recruitment. (A) Peritoneal macrophages were isolated from C57BL/6, IL-152/2,and IL-15 TG mice. Cells were assessed via a NO assay for their ability to respond to LPS with the production of NO at 24 and 48 h. IL-15 TG mice consistently had higher levels of NO production in response to LPS. Representative of four experiments. (B) Lungs were isolated from IL- http://www.jimmunol.org/ 152/2 or C57BL/6 mice 2 d posttumor cell injection (5 3 105). Lungs were digested, and the resultant cells were analyzed via flow cytometry for CD45, F4/80, CD11b, CD11c, and Gr1. Results are reported as percentage of F4/80+ cells. *p , 0.05. identify myeloid cells and various markers to delineate myeloid- derived suppressor cells (CD11b+, Gr1+), lung-resident macro- 2 + + phages (CD11b CD11c ), or recruited macrophages (CD11b FIGURE 8. In the absence of IL-15, CD4 T cells are of a more Th2 by guest on September 25, 2021 Gr12) (43). In IL-152/2 mice, there was an increase in recruited phenotype and promote M2 macrophage polarization. (A and B) CD4 2/2 macrophages brought into the lung (Fig. 7B). Because the mac- T cells were isolated from the of IL-15 , C57BL/6, and IL-15 TG rophages in IL-152/2 mice have an M2 polarization, it is likely mice and stimulated nonspecifically (CD3/CD28) for 48 h. Supernatants were collected and assessed for IL-10 and IL-4 production via ELISA. (C) that these macrophages would assist in the formation of metas- 2 2 2 2 Peritoneal macrophages from C57BL/6, IL-15 / , and IL-15 / CD4- tasis. In prior examinations of this metastasis model, recruitment depleted mice were harvested, plated, and tested for production of NO. of macrophages to the site is very important for efficient meta- ***p , 0.001. static seeding and growth (17, 18). IL-152/2 CD4 T cells appear to have a Th2 phenotype and tumor models. Unfortunately, very few studies have examined may contribute to metastasis by altering macrophage extensively the role of IL-15 in metastasis and its effects on im- polarization mune cells other than NK cells or CD8 T cells. In this study, we To determine the phenotype of the CD4 T cells in our model, CD4 have shown in a breast cancer model of metastasis that IL-15 2/2 T cells were isolated from the spleen of IL-15 and control mice overexpression or IL-15/IL-15Ra treatment greatly decreases the and stimulated nonspecifically for 48 h (Fig. 8A, 8B). The CD4 formation of metastatic sites in the lung, whereas lack of IL-15 Tcellswere.85% pure (data not shown). When analyzed for highly promotes the formation of metastasis. The susceptibility of 2/2 cytokine production, IL-15 CD4 T cells were found to produce IL-152/2 mice to metastasis was observed in both the breast cancer higher levels of both IL-10 and IL-4 (Fig. 8A, 8B). This indicated MT model and a melanoma B16F10 model, thus indicating that the 2/2 that IL-15 CD4 T cells were more Th2 skewed than control CD4 effect is not specific to one particular cell line or tumor type. In IL- 2/2 T cells. To determine whether the CD4 T cells in IL-15 mice 15 TG mice, this protection was found to be dependent on NK cells. were contributing to the M2 macrophage phenotype in these mice, This agrees with earlier studies in which the effects of IL-15 in 2/2 2/2 peritoneal macrophages from IL-15 or CD4-depleted IL-15 metastatic models of colon carcinoma or melanoma are NK cell mice were analyzed for their ability to produce NO. Although dependent, not CD8 T cell dependent (7, 9). We observed that the missing significance, in several experiments it was found that CD4 loss of IL-15, and hence NK cells, from IL-152/2 lungs led to depletion restored the ability of these macrophages to make NO a decrease in type 1 cytokines such as IFN-g, CCL5, and XCL1. 2/2 (Fig. 8C). Therefore, CD4 T cells in IL-15 mice promote tumor These cytokines have been described as the Th1-promoting cyto- metastasis, possibly via their effects on macrophage polarization. kines expressed by activated NK cells in certain infections (40, 41). It is also possible that these molecules can be expressed by Th1 Discussion CD4 or CD8 T cells, as well as macrophages (40, 41, 44). These The ability of IL-15 to affect tumor formation and its effects on molecules can affect many cell types to prevent the formation of both NK cells and CD8 T cells have been well studied in a variety of lung metastasis. For example, in a model of melanoma metastasis, The Journal of Immunology 7 production of IFN-g by lung NK cells was critical for resistance to T cell phenotype is directly due to loss of IL-15 or indirect due to metastasis (45). In addition, IFN-g is known to promote the formation its effects on other cells such as NK cells. As mentioned, the of M1 macrophages and Th1 T cells (46). Chemokines such as CCL5 macrophages in IL-152/2 mice produce very little NO, but when and XCL1 have been found to play a role in antitumor defense by we depleted CD4 T cells they produced wild-type levels of NO. recruiting immune cells such as NK cells, Th1 CD4 T cells, and CD8 Thus, the lack of IL-15 in IL-152/2 mice promotes CD4 T cells CTL into the site of expression, in this case the lung (47–49). Due to that are more Th2 biased and promotes the formation of M2 the significant susceptibility of IL-15 knockout mice to metastasis, it macrophages. It was confirmed that in vivo, CD4 depletion in IL- is likely that the lack of NK cells in these mice could be a significant 152/2 mice reduced the formation of metastasis. Therefore, CD4 contributor to susceptibility. Interestingly, when we depleted NK cells T cells play an important role in promoting metastasis, and this from control mice, it did not fully recapitulate the degree of metas- most likely involves their impact on macrophage polarization. tasisseeninIL-152/2 mice. This indicates that the absence of IL-15 Further examination into whether the effects of IL-15 loss/gain on has more of an effect on metastasis than the loss of NK cells alone. CD4 T cell and macrophage phenotype are direct or indirect When IL-15 is absent, there must be an effect on other factors that are would be of interest. important in promoting metastasis. Indeed, it has been suggested In this model of metastasis, IL-15 acts to decrease metastasis, previously that IL-15 has antitumor activities that are not dependent whereas the absence of IL-15 promotes metastasis. These effects upon NK cells or CD8 T cells (50). are due to NK cells, but also to the effects of IL-15 on other immune It has recently come to light that macrophages not only have cells such as macrophages and CD4 T cells. Because metastasis is a protumoral role at the primary tumor, they are also very important a significant contributor to mortality in breast cancer, this study for the establishment of metastasis (17, 18). They contribute both indicates that IL-15 may be an ideal immunotherapy, and perhaps Downloaded from to the extravasation of the tumor cells and growth at the metastatic its antitumor effects on immune cells are even more widespread site (17, 18). Therefore, we were interested in determining than previously appreciated. whether overexpression or lack of IL-15 has an impact on mac- rophage phenotype. The macrophages that aid in tumor formation Acknowledgments and metastasis have multiple subtypes, but most of the functions We acknowledge Dr. M. Caliguiri for the IL-15 TG mice and the Mayo they exhibit are more of the M2 phenotype (11, 12, 18). Previ- Clinic for the MT cell line. Marianne Chew is acknowledged for technical http://www.jimmunol.org/ ously, there have been reports that IL-15 affects macrophages by assistance. promoting the secretion of proinflammatory cytokines and in- creasing MHCII (26, 28). In this study, we found that peritoneal Disclosures macrophages from IL-15 TG mice have much higher levels of The authors have no financial conflicts of interest. NO production in comparison with wild type, whereas IL-152/2 macrophages produced little to no NO. NO production is typical of M1 macrophages and is a mechanism by which macrophages References can cause tumor cell death (51). Therefore, IL-15 overexpression 1. Ranson, T., C. A. Vosshenrich, E. Corcuff, O. Richard, W. Muller,€ and J. P. Di Santo. 2003. IL-15 is an essential mediator of peripheral NK-cell homeostasis. by guest on September 25, 2021 promotes the formation of M1 macrophages, whereas lack of IL- Blood 101: 4887–4893. 15 changes macrophages into more of an M2 phenotype. It is 2. Carson, W. E., T. A. Fehniger, S. Haldar, K. Eckhert, M. J. Lindemann, C. F. Lai, unknown whether this is a direct effect of IL-15, or an effect due C. M. Croce, H. Baumann, and M. A. Caligiuri. 1997. A potential role for -15 in the regulation of human survival. J. Clin. to how IL-15 affects other cell types. We also examined the my- Invest. 99: 937–943. eloid cell environment in IL-152/2 and control mice 2 d post- 3. Judge, A. D., X. Zhang, H. Fujii, C. D. Surh, and J. Sprent. 2002. /2 15 controls both proliferation and survival of a subset of memory-phenotype tumor cell injections. We found that IL-15 lungs had an CD8(+) T cells. J. Exp. Med. 196: 935–946. increased proportion of recruited macrophages from the circula- 4. Nelson, B. H. 2008. The impact of T-cell immunity on ovarian cancer outcomes. tion. An increase in this population, not lung-resident macro- Immunol. Rev. 222: 101–116. 5. Marrogi, A. J., A. Munshi, A. J. Merogi, Y. Ohadike, A. El-Habashi, O. L. Marrogi, phages, has been shown to aid in the extravasation and and S. M. Freeman. 1997. Study of tumor infiltrating and trans- establishment of lung metastasis (17). forming growth factor-beta as prognostic factors in breast carcinoma. Int. J. It has been suggested that CD4 T cells play a role in polarizing Cancer 74: 492–501. 6. Blum, D. L., T. Koyama, A. E. M’Koma, J. M. Iturregui, M. Martinez-Ferrer, macrophages and promoting metastasis (20, 52). Denardo et al. C. Uwamariya, J. A. Smith, Jr., P. E. Clark, and N. A. Bhowmick. 2008. Che- (20) found that, in a spontaneous mouse breast tumor model, CD4 mokine markers predict biochemical recurrence of prostate cancer following prostatectomy. Clin. Cancer Res. 14: 7790–7797. T cells secrete Th2 cytokines that promote the formation of M2 7. Kobayashi, H., S. Dubois, N. Sato, H. Sabzevari, Y. Sakai, T. A. Waldmann, and macrophages in the primary tumor. Removal of these CD4 T cells Y. Tagaya. 2005. Role of trans-cellular IL-15 presentation in the activation of NK promoted TAMs with a M1 phenotype, and, although this did not cell-mediated killing, which leads to enhanced tumor immunosurveillance. Blood 105: 721–727. affect primary tumor growth, it did decrease the amount of me- 8. Tang, F., L. T. Zhao, Y. Jiang, D. N. Ba, L. X. Cui, and W. He. 2008. Activity of tastasis (20). In our case, we are modeling metastasis by injecting recombinant human interleukin-15 against tumor recurrence and metastasis in MT tumor cells i.v., so we will only observe the effects of mice. Cell. Mol. Immunol. 5: 189–196. 9. Bessard, A., V. Sole´, G. Bouchaud, A. Que´me´ner, and Y. Jacques. 2009. High macrophages/CD4 T cells at the metastatic site. The first step was antitumor activity of RLI, an interleukin-15 (IL-15)-IL-15 receptor alpha fusion to determine whether IL-15 was able to affect the Th1/Th2 bal- protein, in metastatic melanoma and colorectal cancer. Mol. Cancer Ther. 8: 2736–2745. ance of CD4 T cells. It has previously been shown that IL-15 can 10. Martinez, F. O., A. Sica, A. Mantovani, and M. Locati. 2008. Macrophage ac- cause proliferation of CD4 T cells (naive and/or memory), and tivation and polarization. Front. Biosci. 13: 453–461. there are contrasting results as to whether it promotes T regulatory 11. Joyce, J. A., and J. W. Pollard. 2009. Microenvironmental regulation of metas- tasis. Nat. Rev. Cancer 9: 239–252. cells or highly active Th1 CD4 T cells (29, 30, 32). This lack of 12. Mantovani, A., S. Sozzani, M. Locati, P. Allavena, and A. Sica. 2002. Macro- consensus between studies is most likely due to the different phage polarization: tumor-associated macrophages as a paradigm for polarized in vitro and in vivo conditions under which they investigated the M2 mononuclear phagocytes. Trends Immunol. 23: 549–555. 2/2 13. Stout, R. D., C. Jiang, B. Matta, I. Tietzel, S. K. Watkins, and J. Suttles. 2005. effect. We have shown that CD4 T cells isolated from IL-15 Macrophages sequentially change their functional phenotype in response to mice are capable of producing high levels of the Th2 cytokines IL- changes in microenvironmental influences. J. Immunol. 175: 342–349. 14. Sica, A., P. Larghi, A. Mancino, L. Rubino, C. Porta, M. G. Totaro, M. Rimoldi, 10 and IL-4. This indicates a more Th2 CD4 T cell in mice that S. K. Biswas, P. Allavena, and A. Mantovani. 2008. Macrophage polarization in lack IL-15. We have not established whether this change in CD4 tumour progression. Semin. Cancer Biol. 18: 349–355. 8 THE EFFECTS OF IL-15 ON BREAST CANCER METASTASIS

15. Kaplan, R. N., R. D. Riba, S. Zacharoulis, A. H. Bramley, L. Vincent, C. Costa, 33. Ben Ahmed, M., N. Belhadj Hmida, N. Moes, S. Buyse, M. Abdeladhim, D. D. MacDonald, D. K. Jin, K. Shido, S. A. Kerns, et al. 2005. VEGFR1- H. Louzir, and N. Cerf-Bensussan. 2009. IL-15 renders conventional positive haematopoietic bone marrow progenitors initiate the pre-metastatic lymphocytes resistant to suppressive functions of regulatory T cells through niche. Nature 438: 820–827. activation of the phosphatidylinositol 3-kinase pathway. J. Immunol. 182: 16. Hiratsuka, S., A. Watanabe, H. Aburatani, and Y. Maru. 2006. Tumour-mediated 6763–6770. upregulation of chemoattractants and recruitment of myeloid cells predetermines 34. Chow, K. P., J. M. Lee, J. T. Qiu, S. K. Liao, S. C. Lin, S. L. Hsu, N. N. Wu, lung metastasis. Nat. Cell Biol. 8: 1369–1375. Y. F. Lin, and T. S. Wu. 2011. Enhanced IL-10 production by CD4+ T cells 17. Qian, B., and Y. Deng. J. H. Im, R. J. Muschel, Y. Zou, J. Li, R. A. Lang, and primed in IL-15Ra-deficient mice. Eur. J. Immunol. 41: 3146–3156. J. W. Pollard. 2009. A distinct macrophage population mediates metastatic breast 35. Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J. L. Viney, M. Embers, cancer cell extravasation, establishment and growth. PLoS One 4: e6562. N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, et al. 2000. Reversible defects 18. Qian, B. Z., J. Li, H. Zhang, T. Kitamura, J. Zhang, L. R. Campion, E. A. Kaiser, in natural killer and memory CD8 T cell lineages in -deficient L. A. Snyder, and J. W. Pollard. 2011. CCL2 recruits inflammatory monocytes to mice. J. Exp. Med. 191: 771–780. facilitate breast-tumour metastasis. Nature 475: 222–225. 36. Fehniger, T. A., K. Suzuki, A. Ponnappan, J. B. VanDeusen, M. A. Cooper, 19. Tiemessen, M. M., A. L. Jagger, H. G. Evans, M. J. van Herwijnen, S. John, and S. M. Florea, A. G. Freud, M. L. Robinson, J. Durbin, and M. A. Caligiuri. 2001. L. S. Taams. 2007. CD4+CD25+Foxp3+ regulatory T cells induce alternative Fatal leukemia in interleukin 15 transgenic mice follows early expansions in activation of human monocytes/macrophages. Proc. Natl. Acad. Sci. USA 104: natural killer and memory phenotype CD8+ T cells. J. Exp. Med. 193: 219–231. 19446–19451. 37. Rubinstein, M. P., M. Kovar, J. F. Purton, J. H. Cho, O. Boyman, C. D. Surh, and 20. DeNardo, D. G., J. B. Barreto, P. Andreu, L. Vasquez, D. Tawfik, N. Kolhatkar, J. Sprent. 2006. Converting IL-15 to a superagonist by binding to soluble IL- and L. M. Coussens. 2009. CD4(+) T cells regulate pulmonary metastasis of 15Ralpha. Proc. Natl. Acad. Sci. USA 103: 9166–9171. mammary carcinomas by enhancing protumor properties of macrophages. 38. Bergamaschi, C., M. Rosati, R. Jalah, A. Valentin, V. Kulkarni, C. Alicea, Cancer Cell 16: 91–102. G. M. Zhang, V. Patel, B. K. Felber, and G. N. Pavlakis. 2008. Intracellular 21. Chan, T., E. A. Pek, K. Huth, and A. A. Ashkar. 2011. CD4(+) T-cells are im- interaction of interleukin-15 with its receptor alpha during production leads to portant in regulating macrophage polarization in C57BL/6 wild-type mice. Cell. mutual stabilization and increased bioactivity. J. Biol. Chem. 283: 4189–4199. Immunol. 266: 180–186. 39. Srivastava, S., A. Lundqvist, and R. W. Childs. 2008. Natural killer cell im- 22. Savage, N. D., T. de Boer, K. V. Walburg, S. A. Joosten, K. van Meijgaarden, munotherapy for cancer: a new hope. Cytotherapy 10: 775–783. A. Geluk, and T. H. Ottenhoff. 2008. Human anti-inflammatory macrophages 40. Dorner, B. G., A. Scheffold, M. S. Rolph, M. B. Huser, S. H. Kaufmann, Downloaded from induce Foxp3+ GITR+ CD25+ regulatory T cells, which suppress via A. Radbruch, I. E. Flesch, and R. A. Kroczek. 2002. MIP-1alpha, MIP-1beta, membrane-bound TGFbeta-1. J. Immunol. 181: 2220–2226. RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 23. Wang, X., Y. Cui, G. Luo, Q. Wang, J. Hu, W. He, J. Yuan, J. Zhou, Y. Wu, cytokines. Proc. Natl. Acad. Sci. USA 99: 6181–6186. X. Sun, et al. 2012. Activated mouse CD4(+)Foxp3(-) T cells facilitate mela- 41. Dorner, B. G., H. R. Smith, A. R. French, S. Kim, J. Poursine-Laurent, noma metastasis via Qa-1-dependent suppression of NK-cell cytotoxicity. Cell D. L. Beckman, J. T. Pingel, R. A. Kroczek, and W. M. Yokoyama. 2004. Co- Res. 22: 1696–1706. ordinate expression of cytokines and chemokines by NK cells during murine 24. Tan, W., W. Zhang, A. Strasner, S. Grivennikov, J. Q. Cheng, R. M. Hoffman, cytomegalovirus infection. J. Immunol. 172: 3119–3131.

and M. Karin. 2011. Tumour-infiltrating regulatory T cells stimulate mammary 42. Qian, B. Z., and J. W. Pollard. 2010. Macrophage diversity enhances tumor http://www.jimmunol.org/ cancer metastasis through RANKL-RANK signalling. Nature 470: 548–553. progression and metastasis. Cell 141: 39–51. 25. Carson, W. E., M. E. Ross, R. A. Baiocchi, M. J. Marien, N. Boiani, 43. Connelly, L., W. Barham, H. M. Onishko, L. Chen, T. P. Sherrill, T. Zabuawala, K. Grabstein, and M. A. Caligiuri. 1995. Endogenous production of interleukin M. C. Ostrowski, T. S. Blackwell, and F. E. Yull. 2011. NF-kappaB activation 15 by activated human monocytes is critical for optimal production of - within macrophages leads to an anti-tumor phenotype in a mammary tumor lung gamma by natural killer cells in vitro. J. Clin. Invest. 96: 2578–2582. metastasis model. Breast Cancer Res. 13: R83. 26. Alleva, D. G., S. B. Kaser, M. A. Monroy, M. J. Fenton, and D. I. Beller. 1997. 44. Muller,€ K., S. Bischof, F. Sommer, M. Lohoff, W. Solbach, and T. Laskay. 2003. IL-15 functions as a potent autocrine regulator of macrophage proinflammatory Differential production of macrophage inflammatory protein 1gamma (MIP- cytokine production: evidence for differential receptor subunit utilization asso- 1gamma), lymphotactin, and MIP-2 by CD4(+) Th subsets polarized in vitro ciated with stimulation or inhibition. J. Immunol. 159: 2941–2951. and in vivo. Infect. Immun. 71: 6178–6183. 27. Liu, G., Q. Zhai, D. Schaffner, C. Bradburne, A. Wu, A. Hayford, S. Popov, 45. Takeda, K., M. Nakayama, M. Sakaki, Y. Hayakawa, M. Imawari, K. Ogasawara, E. Grene, C. Bailey, and K. Alibek. 2004. IL-15 induces IFN-beta and iNOS K. Okumura, and M. J. Smyth. 2011. IFN-g production by lung NK cells is

gene expression, and antiviral activity of murine macrophage RAW 264.7 cells. critical for the natural resistance to pulmonary metastasis of B16 melanoma in by guest on September 25, 2021 Immunol. Lett. 91: 171–178. mice. J. Leukoc. Biol. 90: 777–785. 28. Ruckert,€ R., K. Brandt, M. Ernst, K. Marienfeld, E. Csernok, C. Metzler, 46. Mantovani, A. 2006. Macrophage diversity and polarization: in vivo veritas. V. Budagian, E. Bulanova, R. Paus, and S. Bulfone-Paus. 2009. Interleukin-15 Blood 108: 408–409. stimulates macrophages to activate CD4+ T cells: a role in the pathogenesis of 47. Lapteva, N., and X. F. Huang. 2010. CCL5 as an adjuvant for cancer immuno- ? Immunology 126: 63–73. therapy. Expert Opin. Biol. Ther. 10: 725–733. 29. Seder, R. A. 1996. High-dose IL-2 and IL-15 enhance the in vitro priming of 48. Huang, H., F. Li, J. R. Gordon, and J. Xiang. 2002. Synergistic enhancement of naive CD4+ T cells for IFN-gamma but have differential effects on priming for antitumor immunity with adoptively transferred tumor-specific CD4+ and CD8+ IL-4. J. Immunol. 156: 2413–2422. T cells and intratumoral lymphotactin transgene expression. Cancer Res. 62: 30. Niedbala, W., X. Wei, and F. Y. Liew. 2002. IL-15 induces type 1 and type 2 2043–2051. CD4+ and CD8+ T cells proliferation but is unable to drive cytokine production 49. Emtage, P. C., Z. Xing, Y. Wan, A. Zlotnik, F. L. Graham, and J. Gauldie. 2002. in the absence of TCR activation or IL-12/IL-4 stimulation in vitro. Eur. J. Adenoviral-mediated gene transfer of lymphotactin to the lungs of mice and rats Immunol. 32: 341–347. results in infiltration and direct accumulation of CD4+, CD8+, and NK cells. J. 31. Geginat, J., F. Sallusto, and A. Lanzavecchia. 2001. Cytokine-driven prolifera- Interferon Cytokine Res. 22: 573–582. tion and differentiation of human naive, central memory, and effector memory 50. Davies, E., S. Reid, M. F. Medina, B. Lichty, and A. A. Ashkar. 2010. IL-15 has CD4(+) T cells. J. Exp. Med. 194: 1711–1719. innate anti-tumor activity independent of NK and CD8 T cells. J. Leukoc. Biol. 32. Benito-Miguel, M., Y. Garcı´a-Carmona, A. Balsa, C. Pe´rez de Ayala, T. Cobo- 88: 529–536. Iba´n˜ez, E. Martı´n-Mola, and M. E. Miranda-Caru´s. 2009. A dual action of 51. Albina, J. E., and J. S. Reichner. 1998. Role of nitric oxide in mediation of rheumatoid arthritis synovial fibroblast IL-15 expression on the equilibrium macrophage cytotoxicity and apoptosis. Cancer Metastasis Rev. 17: 39–53. between CD4+CD25+ regulatory T cells and CD4+CD25- responder T cells. J. 52. Mantovani, A., P. Allavena, A. Sica, and F. Balkwill. 2008. Cancer-related in- Immunol. 183: 8268–8279. flammation. Nature 454: 436–444.