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Immunology |
Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland
Requests for reprints: Suzanne Ostrand-Rosenberg, Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250. Phone: 410-455-2237; Fax: 410-455-3875; E-mail: srosenbe{at}umbc.edu.
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chain when primary tumor is present and that suppress T cells by producing arginase; and (c) production of activated lymphocytes. Macrophages from wild-type BALB/c mice are polarized by interleukin-13 (IL-13) towards a tumor-promoting M2 phenotype, thereby inhibiting the generation of tumoricidal M1 macrophages. In contrast, CD1–/– mice, which are deficient for IL-13 because they lack IL-13–producting NKT cells, generate M1 macrophages that are cytotoxic for 4T1 via the production of nitric oxide. Although tumoricidal macrophages are a necessary component of immune surveillance in CD1–/– mice, they alone are not sufficient for tumor resistance because IL-4R
–/– mice have M1 macrophages and retain high levels of myeloid suppressor cells after surgery; in addition, they are susceptible to 4T1 metastatic disease. These results show that effective immune surveillance against established metastatic disease is negatively regulated by IL-13 and requires the induction of tumoricidal M1 macrophages and lymphocytes combined with a reduction in tumor-induced myeloid suppressor cells. (Cancer Res 2005; 65(24): 11743-51) |
Introduction |
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We (13) and others (8, 9, 14) have reported that CD1–/– mice have enhanced immune surveillance against tumors and have proposed that deletion of the CD1d gene removes an inhibitor that blocks antitumor immunity. In our studies, we have used the spontaneously metastatic BALB/c-derived 4T1 mammary carcinoma (15–17). This tumor closely models human breast cancer in many of its characteristics, including its pattern of metastatic spread (18). Also similar to many human cancers (19–23), 4T1 induces a profound immune suppression, which can be partially reversed if the primary tumor is removed (24). Our finding that CD1–/– mice, whose primary tumors are surgically removed survive indefinitely despite the presence of metastatic disease, has led us to hypothesize that immune surveillance is blocked in wild-type mice by two factors: (a) an inhibitor that is regulated by the CD1d gene and (b) immune suppression induced by primary tumor. Terabe et al., using the 15-12RM fibrosarcoma, have also concluded that wild-type mice contain an inhibitor of immune surveillance and have identified the inhibitor as the cytokine IL-13. They argue that IL-13 blocks immune surveillance by activating Gr1+CD11b+ myeloid-derived suppressor cells (MSC) that secrete the immunosuppressive cytokine transforming growth factor ß (TGFß; refs. 8, 9). Although the immune suppression present in mice with 4T1 primary tumors is also mediated by Gr1+CD11b+ MSC, unlike the 15-12RM tumor system, 4T1-induced MSC are not induced by IL-13 (present report) and do not produce TGFß (25), indicating that resistance to the 15-12RM and 4T1 tumors is mediated by different mechanisms.
Previous studies with the 4T1 tumor in signal transducer and activator of transcription 6–deficient (STAT6–/–) mice showed that in addition to MSC, macrophages also regulate tumor growth (25). Macrophages polarized towards an M2 phenotype, produce arginase, and support tumor growth. In contrast, M1 macrophages, which produce inducible nitric oxide synthase (iNOS), are tumoricidal and mediate tumor regression (26).
It is important to clarify the mechanisms that promote immune surveillance and facilitate tumor regression because a better understanding of these mechanisms may lead to strategies that enhance tumor-specific immunity. Therefore, we have studied the pathways leading to effective immune surveillance against the 4T1 mammary carcinoma in CD1–/–, mice whose primary tumors have been surgically removed but retain disseminated, metastatic disease. We find that effective immune surveillance requires a combination of three conditions: (a) the generation of iNOS-producing tumoricidal M1 macrophages that are produced because CD1–/– mice are deficient for IL-13, which polarizes macrophages to an M2 phenotype; (b) a rapid decrease in the quantity of myeloid-derived Gr1+CD11b+ suppressor cells that are elevated when primary tumor is present and that suppress CD4+ and CD8+ T-cell activation via the production of arginase and reactive oxygen species; and (c) the activation of functional lymphocytes.
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Materials and Methods |
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-deficient (IL-4R–/–) and RAG2-deficient (RAG–/–) mice were from The Jackson Laboratory (Bar Harbor, ME) and Taconic Farms (Germantown, MD), respectively. All strains are on a BALB/c background. Female mice of 8 to 16 weeks were used for all studies. Mice were maintained and/or bred in the University of Maryland Baltimore County (UMBC) animal facility according to the NIH. All animal procedures are approved by the UMBC Institutional Animal Care and Use Committee.
Reagents and antibodies. Sodium thioglycolate and lipopolysaccharide (LPS) were from Difco (Detroit, MI); recombinant mouse IFN
was from Pierce-Endogen (Rockford, IL); dichlorodihydrofluorescein diacetate (DCFDA) and dihydroethidium (DHE) were from Molecular Probes (Eugene, OR). Hemagglutinin (HA) peptide 518-526 and OVA peptide 323-339 were synthesized at the University of Maryland, Baltimore.
Vß8.1,2-PE, CD1d1.1-PE, Gr1-PE, rat IgG2a-PE isotype, and rat IgG2a-FITC isotype were from BD PharMingen (San Jose, CA). CD3-FITC was from Abcam (Cambridge, MA). CD11b-FITC and KJ1-26, an anti-clonotypic monoclonal antibody (mAb) that recognizes the DO11.10 TCR (29), were from Caltag (Burlingame, CA). mAb to arginase 1 and rat anti-mouse Gr-1 antibody for magnetic-affinity cell sorting (MACS; clone RB6-8C5) were from BD PharMingen.
Cell lines, tumor challenges, surgery, and metastasis assay. The J774 macrophage cell line (American Type Culture Collection, Manassas, VA) was maintained in DMEM (Biofluids, Rockville, MD; ref. 25). Mice were inoculated in the abdominal mammary gland with 7,000 4T1 cells, and primary tumor growth and lung metastases were measured (17, 18, 24). Tumor size was measured on the day of surgery, tumor diameter was calculated as the square root of length x width, and primary tumors were surgically removed (30). For experiments comparing non-surgery versus post-surgery groups, mice were inoculated with 4T1 on day 0, and tumor diameters were measured on the day of surgery. Mice were then divided into two groups so that the average tumor diameters for the groups were not significantly different. Primary tumors were removed from one group ("post-surgery") and left in place for the other group ("non-surgery").
T-cell and macrophage depletions. Mice were depleted for CD4+ (mAb GK1.5) or CD8+ (mAb 2.43) T cells or with irrelevant antibodies as described (31). Liposomes loaded with clondronate or control liposomes without clondronate were used to deplete macrophages (32). Briefly, mice were injected i.p. on days 1 and 4 after surgery with 0.2 mL of clodronate or control PBS liposomes and thereafter once a week with 0.1 mL of clodronate or control PBS liposomes. Treatment continued until all of the experimental mice were moribund.
Flow cytometry. Live cells were labeled for cell surface molecules by direct immunofluorescence (18). Samples were analyzed on an Epics XL flow cytometer and analyzed using Expo32 ADC software (Beckman Coulter, Miami, FL).
Myeloid suppressor cells and reactive oxygen species. Splenic MSC were positively purified by magnetic bead sorting using LS columns and rat anti-mouse Gr1 antibody with anti-rat IgG microbeads (ref. 25; Miltenyi Biotec, Auburn, CA). Purifed MSC were assayed by flow cytometry and were >90% Gr1+CD11b+. Reactive oxygen species (ROS) production was measured by DCFDA and DHE (25).
Macrophage assays. Peritoneal macrophages were generated by injecting 1 mL of sterile 3% Brewer thioglycolate medium (Difco) in distilled water i.p. Five days later, mice were euthanized by CO2 asphyxiation, their abdomens were wiped with 70% alcohol, 10 mL of sterile PBS were injected into the peritoneal cavity, and the resulting peritoneal fluid was withdrawn aseptically. Contaminating RBC were lysed with Gey's solution, and the peritoneal exudate cells were washed twice and plated at 1.5 x 106/mL in 0.5 mL DMEM containing 10% FCS in 24-well plates. Nonadherent cells were removed after a 3-hour incubation at 37°C in 5% CO2. The resulting macrophages were activated with IFN and LPS at final concentrations of 2 and 100 ng/mL, respectively, for 16 hours in DMEM, 5% FCS. In some experiments macrophages were stimulated with IL-4 or IL-13 at 50 ng/mL for 16 hours in DMEM containing 5% FCS before their activation with IFN and LPS.
Western blots. Macrophages were washed with excess PBS and resuspended in 200 µL of lysis buffer [one tablet of proteinase inhibitor mix (Roche, Indianapolis, IN), 2 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L sodium orthovanadate, 5% Triton X-100 in 10 mL H2O]. Lysates were microfuged (3,000 x g for 10 minutes at 4°C), the clarified supernatants were electrophoresced in 12% SDS-PAGE gels, and the proteins were blotted onto Hybond-polyvinylidene difluoride membranes (Amersham, Piscataway, NJ) and immunoblotted with mAbs to arginase 1 (33). Proteins were detected using Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).
Nitric oxide and cytotoxicity assays. Nitric oxide (NO) was measured using Griess reagent (34) as described (25). Data are the mean ± SD of triplicate wells. Macrophage cytotoxicity was determined by the procedure of Decker and Lohmann-Matthes (35) as described (25). Values are the average of triplicates ± SD. Background values for media were subtracted from each point. Activated and nonactivated macrophages without 4T1 gave no lactate dehydrogenase release.
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T-cell proliferation assay. T-cell proliferation and transwell experiments were done as described (25). All points were run in triplicates. Data are expressed as:
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CD3 expression. Cells were mixed with peptide and with or without irradiated MSC (5,000 rad) in 24-well plates [5 x 105 T cells, 106 MSC, in 500 µL HL1 culture medium (Bio Whittaker, Walkersville, MD) per well]. After 3 days of culture, cells were harvested, labeled for cell surface markers [KJ1-26-tricolor mAb for D011.10 with CD4-PE; or Vß8-PE mAb for clone 4 with CD8-tricolor (all at a 1:50 dilution)], fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and stained with a 1:20 dilution of CD3-FITC mAb. Labeled cells were analyzed for expression of CD3 by gating on double-positive (CD4+KJ1-26+ or CD8+Vß8+) cells.
Statistical analysis. Student's t test for unequal variance was done using Microsoft Excel 2000.
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Myeloid suppressor cell levels return to normal in CD1-deficient mice after removal of primary tumor. MSC accumulate in some tumor-bearing patients and animals and are potent inhibitors of cell-mediated, tumor-specific immunity (19–23). These cells are immature cells that are in the process of differentiating into mature granulocytes, dendritic cells, or macrophages and are identified by their expression of Gr1 and CD11b. We have previously found that 4T1 tumor progression is associated with the accumulation of MSC (25). To determine if the resistance of CD1–/– mice is related to MSC activity, MSC levels were measured in tumor-bearing CD1–/– and CD1-competent mice. CD1–/– and wild-type BALB/c mice were inoculated with 4T1 tumor cells, and splenocytes were harvested 30 to 39 days later and analyzed for CD11b+ Gr1+ cells (tumor diameter at surgery: BALB/c, 6.05 ± 0.75 mm; CD1–/–, 6.38 ± 0.8 mm). Tumor-free BALB/c and CD1–/– mice have <8% splenic MSC, whereas tumor-bearing (non-surgery) mice have elevated levels of MSC (Fig. 2A; BALB/c, 23 ± 11%; CD1–/–, 26 ± 5%). Therefore, non-surgery CD1–/– and CD1-competent mice both have elevated levels of MSC relative to tumor-free mice.
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The accumulation of MSC is most likely driven by tumor-secreted factors (37). Therefore, the decrease of MSC in post-surgery CD1–/– mice may be due to less metastatic disease in the CD1–/– versus wild-type mice. To test this hypothesis, we plotted the number of metastatic cells versus the percentage of MSC for individual post-surgery mice (Fig. 2B; similar results were obtained for mice with >35% MSC; data not shown). Both BALB/c and CD1–/– mice have extensive metastatic disease, and there is no correlation between percentage of MSC and the number of metastatic cells. Therefore, the decrease in MSC in post-surgery CD1–/– mice is independent of metastatic disease.
Lymphocytes may also play a role in driving MSC levels. To determine if lymphocytes are involved, splenic MSC levels were determined for BALB/c RAG–/– mice inoculated with 4T1 according to the schedule in Fig. 2A (tumor diameter: non-surgery, 5.07 ± 1.2 mm; post-surgery, 5.57 ± 0.95 mm). Likewise, tumor diameter of non-surgery RAG–/–, BALB/c, and CD1–/– mice were similar when MSC levels were measured (RAG–/–, 9.3 ± 1.5 mm; BALB/c, 9.5 ± 0.73; CD1–/–, 10.24 ± 1.10). The baseline level of Gr1+CD11b+ splenocytes in tumor-free RAG–/– mice is <8%, whereas non-surgery RAG–/– mice have significantly (P < 0.01) more MSC than BALB/c or CD1–/– mice (Fig. 2; RAG–/– MSC, 51.8 ± 6%). After surgery, MSC in RAG–/– mice remain significantly higher than in BALB/c or CD1–/– mice (P < 0.01).
To determine if CD4+ and/or CD8+ T cells are involved in resistance, post-surgery CD1–/– mice were in vivo depleted for CD4+ or CD8+ T cells or treated with irrelevant antibodies. Both CD4+ and CD8+ T cells are required for tumor resistance because 100% of CD4-depleted (three of three) and 80% of CD8-depleted (four of five) post-surgery CD1–/– mice but none of the irrelevant antibody treated mice (three of three) die. Therefore, lymphoid cells are essential for tumor rejection and may reduce MSC in post-surgery BALB/c and CD1–/– and mice.
Myeloid suppressor cells inhibit T cells by an arginase-dependent mechanism. CD1–/– mice may have greater tumor immunity because their MSC are less suppressive than MSC of BALB/c mice. To test this possibility, splenocytes from non-surgery BALB/c and CD1–/– mice were MACS purified for Gr1 (>91% and 93% Gr1+CD11b+ for BALB/c and CD1–/–, respectively). The resulting MSC were then cocultured with antigen-specific CD4+ or CD8+ syngeneic T cells or CD4+ allogeneic T cells plus the appropriate peptide (H-2d D011.10 with OVA-peptide, H-2d clone 4 with HA-peptide, or H-2k 3A9 with HEL, respectively), and T-cell activation was measured by [3H]thymidine uptake (Fig. 3A). On a per cell basis, purified BALB/c and CD1–/– MSC equally suppress syngeneic CD4+ or CD8+ or allogeneic CD4+ T cells.
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To determine if suppression requires direct contact between MSC and T cells, CD1–/– MSC were suspended in transwell chambers containing OVA peptide–pulsed DO11.10 T cells (Fig. 3C). Proliferation of DO11.10 cells was not inhibited when the MSC were separated from the T cells by a semipermeable membrane. Therefore, suppression requires direct contact between the MSC and the affected T cells.
Myeloid suppressor cells down-regulate T-cell receptor–associated zeta chain in CD4+ but not CD8+ T cells. Rodriguez et al. (39) and Zabaleta et al. (40) have shown that T-cell dysfunction caused by macrophages or bacteria is associated with the down-regulation of the TCR-associated CD3 chain. To determine if MSC induce suppression by this mechanism, OVA peptide–pulsed CD4+ DO11.10 T cells were cocultured with MSC from BALB/c or CD1–/– mice. Following 3 days of incubation, the cultures were harvested, and the cells were triple labeled for CD3, CD4, and the D011.10 clonotype (KJ1-26). The cells were analyzed by flow cytometry by gating on the DO11.10+ CD4+ double-positive population and assessing CD3 expression. Fifty-three percent of D011.10 transgenic T cells cocultured with OVA peptide have elevated CD3 chain (Fig. 4, top). If BALB/c or CD1–/– MSC are added to the cultures, then only 17% and 15% of the T cells, respectively, have elevated CD3 expression. Therefore, BALB/c and CD1–/– MSC reduce CD3 chain expression, which probably inhibits T-cell activation by inhibiting signal transduction.
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, CD8+ clone 4 T cells were cultured with HA peptide. The resulting cells were gated on the CD8+ Vß8+ double-positive population and analyzed for CD3 expression (Fig. 4, bottom). More than half of the activated CD8+ T cells had elevated CD3. In contrast to CD4+ T cells, CD3 did not decrease following coculture with either BALB/c or CD1–/– MSC. Therefore, BALB/c and CD1–/– MSC suppress CD4+ T cells by down-regulating CD3 chain but suppress CD8+ T cells via a different mechanism. BALB/c and CD1–/– myeloid suppressor cells produce reactive oxygen species. Kusmartsev et al. (41) have shown that ROS are a characteristic of MSC, and we (25) have previously noted that ROS production characterizes different MSC populations. To determine if ROS are differentially expressed in BALB/c versus CD1–/– MSC, splenic MSC were MACS purified from tumor-free and non-surgery mice and analyzed for ROS. Staining with DHE, which measures superoxide, was negative (data not shown). Staining with DCFDA, which measures hydrogen peroxide, hydroxyl radical, peroxynitrile, and superoxide, shows that Gr1+CD11b+ splenic cells from non-surgery BALB/c (Fig. 5A) and CD1–/– (Fig. 5B) mice contain more ROS than MSC from the corresponding tumor-free mice. To assess if arginase is involved in ROS production, the arginase inhibitor nor-NOHA was added to the purified Gr1+CD11b+ cells before their staining with DCFDA. Although nor-NOHA has no effect on ROS expression in CD1–/– MSC, it inhibits ROS expression in BALB/c MSC. Therefore, MSC from both BALB/c and CD1–/– mice contain ROS; however, ROS expression in the CD1–/– MSC is arginase independent, whereas in BALB/c MSC, it is arginase dependent.
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and assayed for iNOS production. LPS- and IFN-activated macrophages from STAT6–/– and IL-4R–/– mice were used as controls. The IL-4R is a common chain that is shared between the receptors for IL-4 and IL-13 and hence is required for transmitting signals for both of these cytokines (46, 47). STAT6 is a transcription factor that transmits signals through the IL-4R (48–50). Therefore, STAT6–/– and IL-4R–/– macrophages should make iNOS regardless of the presence or absence of IL-4 and/or IL-13 (51). Macrophages from all four strains that are activated in vitro in the absence of IL-4 or IL-13 produce iNOS (Fig. 6). However, if the macrophages are treated with IL-4 or IL-13 before activation with LPS and IFN, then BALB/c and CD1–/– macrophages make less iNOS, whereas iNOS production by STAT6–/– and IL-4R–/– is unaffected. Because BALB/c mice produce IL-4 and/or IL-13 in vivo, their macrophages will not make significant levels of iNOS; hence, BALB/c mice will not have M1 macrophages. In contrast, CD1–/– mice will have iNOS-producing M1 macrophages in vivo because they have diminished levels of IL-4 and IL-13 because they lack NKT cells.
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–/– peritoneal macrophages were tested for arginase by Western blot (Fig. 6B). Macrophages were either not activated, activated with LPS plus IFN, pretreated with IL-4 before LPS and IFN activation, pretreated with IL-13 before LPS and IFN activation, unactivated and treated with IL-4, or not activated and treated with IL-13. BALB/c and CD1–/– macrophages, regardless of treatment, contain arginase, whereas IL-4R–/– macrophages contain very little, if any arginase.
Macrophage tumoricidal activity is attributed to iNOS production (26); thus, CD1–/– macrophages may be tumoricidal, although they also contain arginase. To test this hypothesis, BALB/c, CD1–/–, and IL-4R–/– peritoneal macrophages were harvested, activated in vitro with LPS and IFN, and tested for cytotoxic activity against 4T1 cells. CD1–/– and positive control IL-4R–/– macrophages are significantly more cytotoxic than BALB/c macrophages (Fig. 6C; P < 0.05). The cytotoxicity is due to iNOS, because addition of the iNOS inhibitor L-NMMA eliminates the cytotoxic effect, whereas the inactive inhibitor D-NMMA has no effect. Therefore, although CD1–/– macrophages contain both iNOS and arginase, they have strong tumoricidal activity, indicating that they are polarized towards the M1 phenotype. To confirm the role of macrophages in tumor resistance, macrophages were depleted from post-surgery CD1–/– mice by treatment with liposomes loaded with clodronate. Macrophage-depleted (three of three) mice were dead by 42 days after injection of primary tumor, whereas mice treated with PBS loaded liposomes survived (MST > 83 days). Therefore, macrophages are essential for the survival of post-surgery CD1–/– mice.
Interleukin-4 receptor –deficient mice are tumor susceptible and maintain elevated levels of myeloid suppressor cells after surgery. If the presence of M1 macrophages is sufficient for tumor resistance, then IL-4R–/– mice, which have tumoricidal M1 macrophages, may survive after removal of primary tumor. To test this possibility, BALB/c and IL-4R–/– mice were inoculated with 4T1, primary tumors were surgically removed 2 to 3 weeks later, and the mice were followed for survival. IL-4R–/– mice are just as susceptible as BALB/c mice (five of six IL-4R–/– versus seven of eight BALB/c mice die), indicating that despite the presence of M1 macrophages, IL-4R–/– mice do not have heightened tumor immunity. Because MSC decrease to baseline in post-surgery CD1–/– (see Fig. 2) and STAT6–/– (25) mice, we assessed MSC levels in tumor-bearing, non-surgery and post-surgery IL-4R–/– mice (tumor diameter for non-surgery mice: BALB/c, 6.1 ± 1.7 mm; IL-4R–/–, 7.1 ± 1.1 mm; tumor diameter at surgery for the post-surgery groups: BALB/c, 6.5 ± 1 mm; IL-4R–/–, 7.5 ± 0.43 mm). Non-surgery IL-4R–/– mice have elevated MSC (Fig. 7A), and MSC remain elevated after surgery similar to BALB/c (P > 0.05), with only 14% of IL-4R–/– mice having normal levels (<8% MSC). Likewise, post-surgery IL-4R–/– mice contain high levels of metastatic cells (Fig. 7B). Therefore, although IL-4R–/– mice generate tumoricidal M1 macrophages, they are not tumor resistant and have elevated levels of MSC even after removal of primary tumor.
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iNOS-producing M1 macrophages with tumoricidal activity have been described in numerous tumor systems (26, 42, 43). They are cytotoxic because iNOS converts arginine and oxygen to NO, which is toxic. The tumoricidal macrophages of CD1–/– mice are unusual in that they produce iNOS and arginase. Typically, M1 macrophages produce less arginase because it degrades arginine and therefore limits the amount of substrate available for conversion to NO (52, 53). Despite the coexpression of arginase, CD1–/– peritoneal macrophages produce sufficient NO to mediate tumor cell destruction. El-Gayar et al. (53) have shown that IL-13 prevents iNOS production, thereby polarizing macrophages towards a M2 phenotype. Because CD1–/– mice lack NKT cells, which are a major producer of IL-13, it is likely that CD1–/– mice have M1 macrophages because they are deficient for IL-13. This hypothesis is supported by two findings: (a) Addition of IL-13 to cultures of CD1–/– macrophages produces a M2 phenotype; and (b) Macrophages from IL-4R–/– mice, which lack the receptor for IL-13, are M1-type iNOS producers and tumoricidal. Although both IL-13 and IL-4 signal through the IL-4R and STAT6, it is unlikely that IL-4 is the inhibitor of M1 macrophage generation in vivo because IL-4 is produced by activated Th2 cells in addition to NKT cells (54, 55), and CD1–/– mice are only deficient for NKT cells. Therefore, CD1–/– mice, which are deficient for IL-13 (12), constitutively generate iNOS-producing M1 macrophages that are cytotoxic for 4T1 tumor, whereas BALB/c mice produce M2 macrophages under the induction of IL-13.
Previous studies showed that macrophages are essential for immune surveillance against the 4T1 tumor (25, 56). Although M1 macrophages are necessary, their presence is not sufficient for tumor rejection. For example, IL-4R–/– mice, which have tumoricidal M1 macrophages, die from metastatic disease, and CD1–/– mice are only resistant if their primary tumor is removed, although tumoricidal M1 macrophages are present before surgery. In pre-surgery and post-surgery IL-4R–/– mice and in pre-surgery CD1–/– mice, MSC levels are elevated, suggesting that M1 macrophages are ineffective in the presence of large quantities of MSC. In contrast, post-surgery CD1–/– mice with M1 macrophages have baseline levels of MSC and reject metastatic disease. Depletion of M1 macrophages from post-surgery CD1–/– mice makes them susceptible to tumor. Therefore, effective immunity against metastasis requires M1 macrophages coupled with baseline levels of MSC, a condition that only exists in post-surgery CD1–/– mice.
Resistance to 4T1 metastatic disease in CD1–/– mice is reminiscent of resistance to 4T1 in STAT6–/– mice (13, 25, 31). In both strains, tumoricidal M1 macrophages are produced, MSC levels decrease drastically after surgery, and lymphocytes are required. It is likely that IL-13 plays the same role in both strains because IL-13 signaling through the IL4R is via the STAT6/Janus-activated kinase 3 pathway (57). Although IL-13 plays an important role in blocking the production of M1 macrophages, it is not involved in accumulation of MSC or maintaining elevated MSC levels, because non-surgery CD1–/– or IL-4R–/– and post-surgery IL-4R–/– mice have high levels of MSC. Therefore, in addition to their effect on the IL-13/IL4R pathway, CD1 and STAT6 deficiencies also affect another pathway that regulates MSC cell retention.
MSC are present in many patients and experimental animals with cancer and are uniformly immunosuppressive (19–23). Although MSC from different individuals share the ability to suppress, they seem to be a heterogeneous population that suppress via a variety of mechanisms. Down-regulation of the CD3-associated chain and the resulting dysfunction of T cells is a common phenomenon in many patients and experimental mice (reviewed by ref. 58). Rodriguez et al. have shown that such a down-regulation is mediated by macrophages (39, 59). Our findings support this mechanism for the suppression of CD4+ T cells by MSC. However, CD8+ T cells are not down-regulated for CD3 chain in our experiments, suggesting that there are additional mechanisms by which MSC inhibit T-cell activation. Other studies also support the concept that MSC are a functionally heterogeneous population of cells. For example, some MSC inhibit the activation of CD4+ T cells and not CD8+ T cells (60), whereas others inhibit CD8+ T cells and have no effects on CD4+ T cells (20, 22, 61), and others inhibit both CD4+ and CD8+ T cells (ref. 25; current report). The heterogeneity of MSC is further supported by the varied phenotypes that have been reported for these cells. Although many mouse MSC are characterized by their expression of Gr1 and CD11b, other mouse MSC express CD31 and do not express Gr1 and/or CD11b (19). Some MSC express MHC class II, B220, F4/80, CD86, CD16/32, and DEC205 (38), whereas others express MHC class I and do not express MHC class II or costimulatory molecules (20), and others express MHC I and costimulatory molecules but not MHC II (25). Differences also exist in ROS production between the different MSC populations studied. Kusmartsev et al. (41) have shown that ROS production by MSC is arginase dependent. ROS production by the BALB/c MSC described in this report are arginase dependent, whereas ROS production by CD1–/– MSC are arginase independent. These phenotypic differences probably characterize subpopulations of MSC, and it is possible that the different subpopulations have different target cells (e.g., CD4+ versus CD8+ T cells), thereby explaining the functional heterogeneity observed in the different tumor systems.
Others have also shown that CD1–/– mice have enhanced tumor immune surveillance (8, 9, 14, 62), supporting the concept that a deficiency in variant or invariant NKT cells facilitates tumor immunity. Preliminary data with J18–/– mice, which are deficient for invariant NKT cells, indicates that both variant and invariant NKT cells inhibit tumor immunity.1 Terabe et al. also observe that blocking of IL-13 or interfering with signal transduction through the IL4R causes rejection of the CT26 colon carcinoma or block recurrence of the 15-12RM fibrosarcoma. In the 15-12RM fibrosarcoma, IL-13 inhibits tumor immunity by inducing MSC that produce high levels of the immunosuppressive cytokine TGFß (9). However, this mechanism is not responsible for resistance to the 4T1 tumor because (a) 4T1-induced MSC do not produce TGFß (25); (b) IL-4R–/– mice, which are unable to respond to IL-13 because they lack the requisite receptor, are susceptible to 4T1 metastatic disease; and (c) wild-type BALB/c mice treated with an inhibitor of IL-13 remain susceptible (13). Therefore, although we concur that IL-13 is a potent inhibitor of tumor immunity, we find that suppressing IL-13 is not sufficient for generating effective immune surveillance. We also do not agree that IL-13 inhibits immune surveillance by inducing MSC that produce high levels of TGFß, because 4T1-derived MSC do not contain TGFß (25). Our data indicate that IL-13 counteracts immune surveillance by polarizing macrophages away from a tumoricidal M1 phenotype. Although these differences in interpretation of IL-13 function may be due to differences in the tumor systems used, they may also indicate that IL-13 is a pleiomorphic cytokine that blocks immune surveillance via multiple mechanisms.
These studies show that immune surveillance can eliminate metastatic disease in a post-surgery setting. Although effective immunity is a complex process that requires the activation of multiple effector cells (macrophages and lymphocytes) coupled with the down-regulation of suppressive/inhibitory cells (MSC), a better understanding of these mechanisms may reveal strategies for facilitating tumor immunity and extending survival.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. L. van Kaer (Vanderbilt), Bill Wade (Dartmouth), and Ephraim Fuchs (Johns Hopkins) for providing breeding pairs of the CD1–/–, 3A9, and clone 4 mice, respectively, and Sandra Mason for giving excellent care to our mouse colony.
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Received 1/ 6/05. Revised 8/16/05. Accepted 9/ 7/05.
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and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 2001;410:1107–11.[CrossRef][Medline] dependent. J Immunol 2002;169:5796–804.. Cancer Immunol Immunother 2000;49:34–45.[CrossRef][Medline] chain in T lymphocytes. J Immunol 2003;171:1232–9.-chain (CD3). J Immunol 2004;173:586–93. chain between receptors for IL-2 and IL-4. Science 1993;262:1874–7. chain: a functional component of the interleukin-4 receptor. Science 1993;262:1880–3.-dependent phagocytic cells are a critical component of innate immunity against metastatic mammary carcinoma. Cancer Res 2002;62:4406–12.-chain downregulation: curtailing an excessive inflammatory immune response. Nat Rev Immunol 2004;4:675–87.[CrossRef][Medline]This article has been cited by other articles:
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  E. M. Hanson, V. K. Clements, P. Sinha, D. Ilkovitch, and S. Ostrand-Rosenberg Myeloid-Derived Suppressor Cells Down-Regulate L-Selectin Expression on CD4+ and CD8+ T Cells J. Immunol., July 15, 2009; 183(2): 937 - 944. [Abstract] [Full Text] [PDF]
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E. Eruslanov, S. Kaliberov, I. Daurkin, L. Kaliberova, D. Buchsbaum, J. Vieweg, and S. Kusmartsev Altered Expression of 15-Hydroxyprostaglandin Dehydrogenase in Tumor-Infiltrated CD11b Myeloid Cells: A Mechanism for Immune Evasion in Cancer J. Immunol., June 15, 2009; 182(12): 7548 - 7557. [Abstract] [Full Text] [PDF]
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 S. K. Bunt, V. K. Clements, E. M. Hanson, P. Sinha, and S. Ostrand-Rosenberg Inflammation enhances myeloid-derived suppressor cell cross-talk by signaling through Toll-like receptor 4 J. Leukoc. Biol., June 1, 2009; 85(6): 996 - 1004. [Abstract] [Full Text] [PDF]
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S. Ostrand-Rosenberg and P. Sinha Myeloid-Derived Suppressor Cells: Linking Inflammation and Cancer J. Immunol., April 15, 2009; 182(8): 4499 - 4506. [Abstract] [Full Text] [PDF]
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 T. Hagemann, S. K. Biswas, T. Lawrence, A. Sica, and C. E. Lewis Regulation of macrophage function in tumors: the multifaceted role of NF-{kappa}B Blood, April 2, 2009; 113(14): 3139 - 3146. [Abstract] [Full Text] [PDF]
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 A. Tyagi, R. P. Singh, K. Ramasamy, K. Raina, E. F. Redente, L. D. Dwyer-Nield, R. A. Radcliffe, A. M. Malkinson, and R. Agarwal Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-Associated Cytokines and Nuclear Factor-{kappa}B and Signal Transducers and Activators of Transcription 3 Cancer Prevention Research, January 1, 2009; 2(1): 74 - 83. [Abstract] [Full Text] [PDF]
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 S. Donnelly, C. M. Stack, S. M. O'Neill, A. A. Sayed, D. L. Williams, and J. P. Dalton Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages FASEB J, November 1, 2008; 22(11): 4022 - 4032. [Abstract] [Full Text] [PDF]
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S. Watanabe, K. Deguchi, R. Zheng, H. Tamai, L.-x. Wang, P. A. Cohen, and S. Shu Tumor-Induced CD11b+Gr-1+ Myeloid Cells Suppress T Cell Sensitization in Tumor-Draining Lymph Nodes J. Immunol., September 1, 2008; 181(5): 3291 - 3300. [Abstract] [Full Text] [PDF]
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 R. M. Strieter What Differentiates Normal Lung Repair and Fibrosis?: Inflammation, Resolution of Repair, and Fibrosis Proceedings of the ATS, April 15, 2008; 5(3): 305 - 310. [Abstract] [Full Text] [PDF]
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E. Ambrosino, M. Terabe, R. C. Halder, J. Peng, S. Takaku, S. Miyake, T. Yamamura, V. Kumar, and J. A. Berzofsky Cross-Regulation between Type I and Type II NKT Cells in Regulating Tumor Immunity: A New Immunoregulatory Axis J. Immunol., October 15, 2007; 179(8): 5126 - 5136. [Abstract] [Full Text] [PDF]
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B. Zhu, Y. Bando, S. Xiao, K. Yang, A. C. Anderson, V. K. Kuchroo, and S. J. Khoury CD11b+Ly-6Chi Suppressive Monocytes in Experimental Autoimmune Encephalomyelitis J. Immunol., October 15, 2007; 179(8): 5228 - 5237. [Abstract] [Full Text] [PDF]
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P. Loke, I. Gallagher, M. G. Nair, X. Zang, F. Brombacher, M. Mohrs, J. P. Allison, and J. E. Allen Alternative Activation Is an Innate Response to Injury That Requires CD4+ T Cells to be Sustained during Chronic Infection J. Immunol., September 15, 2007; 179(6): 3926 - 3936. [Abstract] [Full Text] [PDF]
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 C. M. Paulos, A. Kaiser, C. Wrzesinski, C. S. Hinrichs, L. Cassard, A. Boni, P. Muranski, L. Sanchez-Perez, D. C. Palmer, Z. Yu, et al. Toll-like Receptors in Tumor Immunotherapy Clin. Cancer Res., September 15, 2007; 13(18): 5280 - 5289. [Abstract] [Full Text] [PDF]
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D. Yang, N. ud Din, D. D. Browning, S. I. Abrams, and K. Liu Targeting Lymphotoxin {beta} Receptor with Tumor-Specific T Lymphocytes for Tumor Regression Clin. Cancer Res., September 1, 2007; 13(17): 5202 - 5210. [Abstract] [Full Text] [PDF]
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P. Sinha, V. K. Clements, S. K. Bunt, S. M. Albelda, and S. Ostrand-Rosenberg Cross-Talk between Myeloid-Derived Suppressor Cells and Macrophages Subverts Tumor Immunity toward a Type 2 Response J. Immunol., July 15, 2007; 179(2): 977 - 983. [Abstract] [Full Text] [PDF]
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 C. Aspord, A. Pedroza-Gonzalez, M. Gallegos, S. Tindle, E. C. Burton, D. Su, F. Marches, J. Banchereau, and A. K. Palucka Breast cancer instructs dendritic cells to prime interleukin 13-secreting CD4+ T cells that facilitate tumor development J. Exp. Med., May 14, 2007; 204(5): 1037 - 1047. [Abstract] [Full Text] [PDF]
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S. K. Watkins, N. K. Egilmez, J. Suttles, and R. D. Stout IL-12 Rapidly Alters the Functional Profile of Tumor-Associated and Tumor-Infiltrating Macrophages In Vitro and In Vivo J. Immunol., February 1, 2007; 178(3): 1357 - 1362. [Abstract] [Full Text] [PDF]
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 D. I. Gabrilovich, V. Bronte, S.-H. Chen, M. P. Colombo, A. Ochoa, S. Ostrand-Rosenberg, and H. Schreiber The Terminology Issue for Myeloid-Derived Suppressor Cells Cancer Res., January 1, 2007; 67(1): 425 - 425. [Full Text] [PDF]
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, individual mouse. From two independent experiments. C, BALB/c or CD1–/– splenocytes or 4T1 cells were stained with FITC-conjugated CD1d mAb (filled histograms) or isotype-matched control mAb (open histograms) and analyzed by flow cytometry. From one of two independent experiments.
28 (post-surgery groups). Tumor-free groups were not given 4T1. On day 
, individual mouse.