MUC1/sec-Expressing Tumors Are Rejected In Vivo by a T Cell-Dependent Mechanism and Secrete High Levels of CCL2¹

Mucin 1 (MUC1)³ is a large, heavily glycosylated transmembrane protein consisting of an extracellular region and transmembrane and cytoplasmic domains (1). Within the extracellular domain are tandem repeats that consist of blocks of 20 aa repeated anywhere from 60–100 times within the same protein. These repeats are normally heavily glycosylated, with many O-linked glycosylation sites. The cytoplasmic portion of MUC1 contains tyrosine phosphorylation sites that may serve as docking sites for transcriptional regulator proteins. The SOS/Grb2 adaptor complex has been shown to bind the phosphorylated MUC1 cytoplasmic domain, leading to activation of the Ras protein, which, in turn, may lead to cell proliferation and survival signaling (2). MUC1 is classically expressed on the apical surface of mucosal epithelial cells; however, recent data point to cells of hemopoetic lineages, such as T (3), B (4), and dendritic cells (5), also expressing MUC1. Functionally, MUC1 is believed to protect the surface of cells from damage and to mediate cell-cell interactions (6).

Cells that have undergone neoplastic transformation often display altered phenotypes, including the expression of chimeric proteins such as BCR-ABL (7) or the overexpression of normal proteins. MUC1 is a classic example of a surface protein that is altered during transformation (8, 9). Not only do tumor cells overexpress the MUC1 protein, but instead of being localized, it is displayed along the entire surface of the cell. The glycosylation status of MUC1 is also affected in tumors. The O-glycosylated core of tumor-associated MUC1 tends to be shorter and less branched, and contains higher amounts of sialic acid than MUC1 found on normal cells (6).

Several alternative splice variants of MUC1 have been identified, including the secreted isoform, MUC1/sec (10). Characteristic of many secreted proteins, MUC1/sec lacks the cytoplasmic and transmembrane regions of MUC1. The function of MUC1/sec has not yet been elucidated, although it has been shown to interact with another cell surface protein capable of signaling, MUC1/Y (11).

In this report we demonstrate that MUC1/sec-expressing cells fail to grow in mice, and that T lymphocytes, in conjunction with cells of the innate immune system, are necessary for rejection of DA-3/sec tumors. We also show that MUC1/sec-expressing cells secrete the chemokine CCL2, which may function to modulate both the innate and adaptive immune responses.

BALB/c mice (H-2^d) between 8 and 12 wk of age were bred in our animal facility at University of Miami. BALB/c^nu/nu (nude), IFN-γ^−/−, CD1^−/−, and C.B6-CMV1^r/Uwa mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C.B6-CMV1^r/Uwa have a BALB/c background with the exception of the NK locus, which is of C57BL/6 origin (12). Animal care and use were performed according to the guidelines of the National Institutes of Health. The DA-3 mammary tumor cell line was developed in our laboratory and maintained as previously described (13). The DA-3/TM, DA-3/neo, and DA-3/sec transfected cell lines were developed in collaboration with I. Keydar and D. Wreschner, Tel Aviv University. The MUC1/TM and MUC1/sec cDNA sequences were cloned in the pCL642 expression vector, and DA-3 cells were transfected as previously described (10, 11, 14). For tumor challenge, cells were trypsinized, washed, resuspended in PBS, and implanted s.c. into mice. Tumor size is reported as the length plus the width of the tumor as measured by calipers.

Depletion of T cell subsets was as follows: BALB/c mice were treated with 100 μg of anti-CD4 (GK1.5) or 200 μg of anti-CD8 (2.43) mAbs before s.c. injection of tumor cell lines. Two hundred microliters of anti-asialo-GM1 (Wako Chemicals, Richmond, VA) Ab was injected into mice i.p. to deplete NK cells. All Abs were administered by i.p. injection at 3 and 1 days before tumor challenge and every 7 days thereafter. Ab treatment led to selective depletion of the targeted subset and was typically >95% as determined by FACS analysis of splenocytes. Similarly, C.B6-CMV1^r/Uwa mice were depleted of NK cells by treatment with 200 μg of anti-NK1.1 (PK136) 5, 3, and 1 day before challenge and continuing every 7 days thereafter for 4 wk.

BALB/c mice were inoculated i.p. with 1 mg of type IV carrageenan (Sigma-Aldrich, St. Louis, MO) 3 and 1 days before challenge and twice a week thereafter to inactivate macrophages (15, 16).

Splenocytes from normal mice or mice injected with 10⁶ tumor cells 14 days before culture were harvested, washed, and counted. In a six-well plate, 10 × 10⁶ splenocytes were seeded with 2 × 10⁵ mitomycin C-treated stimulator cells or medium alone for 5 days at 37°C in 5% CO₂ in RPMI 1640 containing 2 mM l-glutamine, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 10 mM HEPES, and 10% FBS (complete medium). Stimulators consisted of mitomycin C-treated tumor cells. The same tumor cells that were injected into mice were used as in vitro stimulators.

CD8⁺ responders were tested for their cytolytic activity in a standard 4-h ⁵¹Cr release assay. Tumor cells were labeled with 100 μCi of sodium ⁵¹Cr (PerkinElmer, Boston, MA), extensively washed, resuspended in complete medium, and used as target cells (10,000 targets/well) at the indicated E:T cell ratios. The percentage of specific ⁵¹Cr release was calculated as follows: [(mean experimental cpm − mean spontaneous cpm)/(mean maximum cpm − mean spontaneous cpm)] × 100%. Spontaneous release was <15% of maximum release.

Eight- to 12-wk-old BALB/c mice were injected i.p. with 4 × 10⁶ DA-3/sec or DA-3/TM cells resuspended in 0.5 ml of PBS, with control animals receiving injections of PBS alone. Alternatively, mice were injected with 1.5 ml of 3-day serum-free DA-3/sec or DA-3/TM supernatants. Twenty-four hours postinjection, mice were killed, and the peritoneum was washed twice with 10 ml of cold PBS/5 mM EDTA. Exudates were centrifuged, washed with PBS/5 mM EDTA, and counted, and one million cells were stained with F4/80, GR-1, anti-CD3, or isotype control Abs (BD Pharmingen, San Diego, CA) to determine cell phenotypes by analysis on a BD Pharmingen LSR Analyzer.

T cells were purified from the spleens of naive mice or mice s.c. challenged for 2 wk with 10 × 10⁶ DA-3/sec cells using anti-Thy1.2 Abs fused to magnetic beads as described previously (13). Twenty-four hours before T cell purification, naive mice were injected i.p. with 4 × 10⁶ DA-3/sec cells to recruit peritoneal cells. Peritoneal cells were harvested as described above, and 3 × 10⁵ cells were seeded in 24-well plates. After a 2-h incubation, nonadherent cells were removed. Naive or DA-3/sec-primed T cells (7.5 × 10⁵ or 1.5 × 10⁶) were incubated with medium, 5 μg/ml Con A, or 3 × 10⁵ peritoneal cells for 96 h, and supernatants were harvested and used in an IFN-γ cytokine ELISA.

Equal numbers of 3-day confluent tumor cells were harvested for their RNA using Tri-Reagent (Molecular Research Center, Cincinnati, OH). RNA was used for RT-PCR and RNA gene arrays as described previously (13). Briefly, 1 μg of RNA was reverse transcribed, and the cDNA was used as a template for the PCR. PCR conditions for CCL2 and actin consisted of 94°C for 10 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension for 10 min at 72°C. The primer for CCL2 and actin were described previously (13).

The relative mRNA expression of mouse inflammatory cytokine and cytokine receptors was analyzed with the chemiluminescent GEArray Q series (Superarray, Bethesda, MD) according to the manufacturer’s protocol. Briefly, 5 mg of total RNA from each sample was reverse transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) in the presence of biotin-16-dUTP (Roche, Indianapolis, IN). The resulting cDNA probes were hybridized to cDNA fragments spotted on GEArray nylon membranes. The relative expression level of each gene was analyzed using GEArray Analyzer and ScanAlyze software.

CCL2 or IFN-γ present in the supernatants of in vitro cell cultures was measured by the OPTEIA mouse CCL2 or IFN-γ set (BD Pharmingen) according to the manufacturer’s instructions. Absorbance at 450 nm was read on a Tecan SLT Rainbow Reader (Labinstruments, Research Triangle Park, NC), and OD values of samples were converted to picograms per milliliter against a standard curve of known quantities of rmCCL2 or rmIFN-γ.

Data obtained from injecting tumors into nude mice were analyzed using Student’s t test. Comparison of means was performed to determine whether significant differences were observed using ANOVA.

DA-3 cells transfected with the transmembrane form of MUC1 (DA-3/TM), the secreted isoform MUC1/sec (DA-3/sec), or vector only (DA-3/neo) were tested for their ability to form tumors in BALB/c mice. The s.c. implantation of 10⁶ DA-3, DA-3/neo, or DA-3/TM cells formed palpable tumors after 1 wk (Fig. 1,A). These tumors grew progressively at the injection site and necrosed after ∼3 wk. After the establishment of DA-3, DA-3/neo, or DA-3/TM tumors, mice developed metastasis of tumor cells to the lung (data not shown). Interestingly, no tumor was observed at the injection site of 10⁶ DA-3/sec cells in immunocompetent BALB/c mice. Increasing numbers of DA-3/sec cells were injected to determine whether a higher concentration of cells would result in tumor growth (Fig. 1 B). No progressive DA-3/sec tumor formation occurred even after injection of 20 × 10⁶ cells. Twenty million cells do, however, cause the initial growth of a DA-3/sec tumor, which was eradicated by 10 days. The rejection of DA-3/sec tumor cells left the surrounding tissue intact and mice remained healthy with no signs of metastasis up to 9 mo later. In vitro, DA-3/sec cells grow well, with slightly faster growth kinetics than DA-3, DA-3/neo, or DA-3/TM cells. We also observed that DA-3/sec supernatant is nontoxic to other cell types (data not shown); therefore, an immunological basis for rejection of DA-3/sec tumor cells was sought.

The rejection of tumors is a complex process involving recruitment and activation of APCs, subsequently resulting in the activation of effector cells, including T lymphocytes, which have been shown to be important in the clearance of many tumor types (17). To test whether T lymphocytes were involved in the rejection of DA-3/sec cells, 10⁶ DA-3/neo, DA-3/TM, or DA-3/sec cells were implanted s.c. into BALB/c athymic nude mice, which lack a majority of T lymphocytes (Fig. 2,A). DA-3/neo, DA-3/TM, and DA-3/sec cells all formed viable tumors in these T cell-deficient animals. However, DA-3/sec cells had a delayed onset of growth compared with DA-3/neo and DA-3/TM during early tumor formation, suggesting early control of DA-3/sec tumors by a T lymphocyte-independent mechanism (discussed below). These results indicate that the DA-3/sec cells are capable of forming progressively growing tumors and that, in immunocompetent BALB/c animals, T lymphocytes are involved in the rejection of DA-3/sec cells. To characterize which subset of T lymphocytes is necessary for the rejection of DA-3/sec cells, mice were depleted of either CD4 or CD8 subsets. Eight- to 12-wk-old BALB/c mice were injected i.p. with anti-CD4, anti-CD8, or both anti-CD4 and anti-CD8 as described in Materials and Methods. Each mouse within these three groups then received an s.c. injection of 10 × 10⁶ DA-3/sec cells. As indicated in Fig. 2,B, animals depleted of CD4⁺ cells are protected against progressive growth of DA-3/sec tumors, although 100% of the challenged mice initially developed very small nodes at the injection site. These tumors all regressed within 28 days, and the animals remained healthy thereafter, with no signs of metastasis. Animals depleted of CD8⁺ cells showed only partial susceptibility to tumor challenge (Fig. 2,C), as 50% of the challenged CD8-depleted mice did not develop tumors. However, of the animals that showed tumor growth, 30% developed DA-3/sec tumor nodes that then regressed, and 20% developed progressive tumors. Similar to the nude mice, 100% of the animals depleted of both CD4 and CD8 cells failed to be protected against DA-3/sec cell challenge and developed progressively growing tumors (Fig. 2 D). Therefore, T lymphocytes appear to be the major mechanism for the rejection of DA-3/sec cells in vivo.

We demonstrated that the DA-3/neo, DA-3/TM, and DA-3/sec tumor cells form progressively growing tumors in nude mice (Fig. 2,A). However, there is a significant difference in the growth kinetics of DA-3/TM cells and DA-3/neo cells compared with DA-3/sec cells in nude mice, with the appearance of DA-3/sec tumors arising more slowly than that of tumors from the other cell lines. These results suggest a potential role for one or more components of the innate response in slowing the initial DA-3/sec tumor formation. To characterize which cell types might be responsible for early suppression of DA-3/sec growth, we used in vivo effector cell depletions and knockout mice. Eight- to 12-wk-old BALB/c mice were injected i.p. with anti-asialo-GM1 to deplete NK cells. Anti-NK1.1 Ab derived from the PK136 hybridoma efficiently depletes both NK and NKT cells in C57BL/6 mice (18). To use this Ab, BALB/c mice congenic for the C57BL/6 NK locus (12) were obtained. To further assess the effects of NKT cells on preventing DA-3/sec tumor formation, CD1d^−/− mice were used. CD1d is an MHC-like protein that is necessary for the development and activation of NKT cells, and mice lacking this protein fail to develop functional NKT cell populations (19). Each mouse was injected with 10 × 10⁶ DA-3/sec cells s.c., and tumor formation was observed daily. The results demonstrate that NK cells (Fig. 3,A) and NKT cells (Fig. 3, B and C) participate in the early suppression of DA-3/sec tumor cell growth, but tumors fail to progress and, instead, regress after ∼2 wk. The DA-3/sec tumors that form in these mice are all relatively small compared with tumors that progress in the nude or CD4⁺/CD8⁺ depleted mice. The cytokine IFN-γ has been shown to be important in the activation of NK cells and macrophages, polarizes T lymphocytes toward a Th1 response, and is involved in protection against the development of tumors (20). One hundred percent of BALB/c IFN-γ^−/− mice challenged with 10 × 10⁶ DA-3/sec cells developed tumors by day 7, with full regression by day 18 (Fig. 3 D). These results indicate that IFN-γ is important in early DA-3/sec rejection, but is not essential for the permanent rejection of DA-3/sec cells.

The role of macrophages in the rejection of DA3/sec cells was also investigated. BALB/c mice, treated with carrageenan to inactivate macrophages, were injected with 10 × 10⁶ DA-3/sec cells (Fig. 4). DA-3/sec cells formed regressive tumors in macrophage-inactivated mice similar to those seen in the NK-, NKT-, and IFN-γ-deficient mice, suggesting there is also participation by macrophages in early DA-3/sec rejection.

Interestingly, the time of tumor appearance and regression varied depending on the cell type removed (Table I). The data demonstrate that mice lacking NKT cells develop DA-3/sec tumors at 3 days, followed by macrophage- and IFN-γ-deficient animals at 5 days, and finally by NK-depleted mice at 7 days. Overall, these results indicate that early control of the DA-3/sec tumor is mediated in part by NK, NKT, macrophages, and the cytokine IFN-γ.

The in vivo depletion experiments demonstrated conclusively that T lymphocytes are necessary for the prevention of progressive DA-3/sec tumors (Fig. 2). Effector cytotoxic CD8 T lymphocytes mediate the rejection of a virally infected cells and tumor cells in an Ag-specific fashion (21). Therefore, we looked for the presence of DA-3/sec-specific CTL in DA-3/sec-challenged mice. One million DA-3/sec cells were injected s.c. into BALB/c mice, and 2 wk after challenge, splenocytes were harvested and stimulated in vitro for 5 days with mitomycin C-treated DA-3/sec cells. A 4-h cytotoxicity assay was performed using ⁵¹Cr-labeled DA-3, DA-3/TM, DA-3/neo, or DA-3/sec cells as targets. A robust response to DA-3/sec target cells was generated (Fig. 5 A). In contrast, CTL-mediated lysis against DA-3, DA-3/TM, or DA-3/neo cells was not observed. Mice challenged with DA-3, DA-3/TM, or DA-3/neo failed to develop CTL responses against their respective tumor cells (data not shown).

The ability of NKT cells to provide help for the generation of CTL has recently been demonstrated (22). Because NKT cells play an important role in the early rejection of DA-3/sec cells (Fig. 3, B and C), CD1d^−/− mice were tested for their ability to generate DA-3/sec-specific CTL compared with wild-type mice. CTL derived from CD1d^−/− mice had a diminished ability to lyse DA-3/sec cells compared with normal splenocytes (Fig. 5 B). The reduced ability of NKT-deficient mice to generate an immune response is clearly not a factor in preventing DA-3/sec rejection, because no progressive tumors are seen in CD1d^−/− mice. However, these results indicate that NKT cells are able to provide help in the generation of DA-3/sec-specific CTL, possibly via cytokine secretion.

Recruitment of APCs and effector cells to the site of tumor is a major first step in the elimination of tumor. Because DA-3/sec cells are unable to grow in wild-type mice, and this failure to grow is a direct result of both innate and adaptive immune responses, we questioned whether the DA-3/sec cells themselves preferentially recruit host lymphocytes. Eight- to 12-wk-old BALB/c mice were injected i.p. with 4 × 10⁶ DA-3/sec or DA-3/TM cells. After 24 h, the peritoneal cavity was flushed, and the exudates were stained with multiple Abs to determine which cell types were present (Table II). The majority of cells present in the exudates from DA-3/TM-injected mice were GR-1^high cells (73%), indicative of neutrophils (23). In contrast, DA-3/sec cells injected i.p. preferentially recruited F4/80⁺ and Gr-1^low cells, indicative of macrophages (23). Other cell types tested for include lymphocytes (CD3) and dendritic cells (CD11c/CD11b). However, no significant differences were observed in the recruitment of these cells. The recruitment of monocytes/macrophages may be due to a secreted factor, such as a chemokine. To test this, 1.5 ml of serum-free supernatants from equal numbers of DA-3/TM and DA-3/sec cells grown in culture for 3 days were used in place of tumor cells. These experiments gave results similar to those seen with whole cell injections (Table II). This suggests that DA-3/sec cells secrete a soluble factor(s) capable of recruiting inflammatory cell types.

The characterization of the DA-3/sec-recruited peritoneal cells revealed that a large percentage of cells were monocytes/macrophages. Therefore, we next determined whether the DA-3/sec-recruited peritoneal cells were capable of stimulating T cells to produce the cytokine IFN-γ. To accomplish this, purified T cells from naive or DA-3/sec-challenged mice were incubated with peritoneal exudates recruited after 24-h i.p. DA-3/sec cell injections. The results in Table III demonstrate that T cells from both naive and DA-3/sec-challenged mice are stimulated to produce IFN-γ in response to DA-3/sec elicited peritoneal cells. The DA-3/sec-primed T cells respond more efficiently to DA-3/sec-derived peritoneal cells, as IFN-γ production is 2- to 3-fold higher than that of naive mice similarly stimulated.

Because DA-3/sec cells secrete a factor(s) that recruits macrophages in vivo, we performed a cDNA array on total RNA isolated from DA-3 and DA-3/sec cells for inflammatory cytokines/cytokine receptors. DA-3 and DA-3/sec cells were grown to confluence in flasks for 3 days. Cells were then trypsinized and counted, and equal numbers of DA-3 and DA-3/sec cells were lysed. Total RNA was purified and used as a template for the synthesis of cDNA probes. The nonradioactive cDNA was hybridized to the membrane containing 96 genes, washed, and exposed to film according to the manufacturer’s protocol. The results indicate that the greatest and most consistent difference in gene expression between DA-3 and DA-3/sec cells was RNA for the chemokine CCL2 (Fig. 6,A). Normalization of the two blots revealed a 33-fold increase in the CCL2 signal from DA-3/sec compared with DA-3. Another gene, LIX, a neutrophil chemokine (24) was down-regulated ∼2-fold in DA-3/sec cells compared with DA-3 cells. A comparison of DA-3/TM and DA-3/sec RNA was also performed using the inflammatory cytokine/cytokine receptor array. The results also show a similar increase in CCL2 in DA-3/sec cells compared with DA-3/TM (data not shown). To validate the up-regulated expression of CCL2 in DA-3/sec cells, total RNA from DA-3, DA-3/TM, and DA-3/sec cells cultured for 3 days was used for RT-PCR of the CCL2 and actin genes (Fig. 6,B). Independent results from the RT-PCR confirm a dramatic up-regulation of the CCL2 message in DA-3/sec cells compared with DA-3 and DA-3/TM cell lines. The RT-PCR from the DA-3 and DA-3/TM RNA did reveal a small amount of CCL2 expression compared with DA-3/sec. To determine whether there was a difference at the protein level, CCL2 ELISAs were performed on supernatants from counted DA-3, DA-3/TM, DA-3/neo, and DA-3/sec cells of 3-day cultures (Fig. 6 C). A second transfectant of MUC1/sec in DA-3 cells (DA-3/sec22) was also tested for the production of CCL2. These results show that MUC1/sec-expressing cells produce ∼20 ng/ml CCL2/million cells, whereas DA-3, DA-3/neo, and DA-3/TM cells secreted undetectable amounts. A similar amount of CCL2 can be detected by DA-3/sec and DA-3/sec22 cells as early as 20 h after seeding (data not shown), suggesting that CCL2 production is a rapid event. Overall, we show that CCL2 is induced in MUC1/sec transfectants at both RNA and protein levels.

The role that tumor-associated Ags play in the progression and development of an immune response to tumors varies, with some inducing a strong response leading to rejection and others leading to a tolerogenic state, allowing the tumor to successfully grow (25). The transmembrane form of MUC1 is an abundantly expressed glycoprotein in several tumor types and has been shown to enhance the tumorigenicity of neoplasms and negatively regulate the immune response by preventing T cell proliferation and activation (26, 27). MUC1 has several alternatively expressed isoforms, and the effects of these on tumor progression have not, to date, been addressed. The present study was designed to investigate the role that one such variant, MUC1/sec, plays in tumor formation.

We have found that the expression of MUC1/sec in a murine mammary adenocarcinoma cell line, DA-3, renders it unable to grow in wild-type mice. Using nude and T cell-depleted mice, we have also demonstrated there is an immunological basis for this rejection and an absolute necessity for the presence of T lymphocytes in the clearance of DA-3/sec tumor cells (Fig. 2). As the data indicate, DA-3/sec tumors, when implanted into CD4⁺ cell-depleted mice, form small nodes at the site of injection that eventually all regress. When CD8⁺ cells are depleted, a 20% progressive DA-3/sec tumor occurrence is observed. When both CD4⁺ and CD8⁺ are eliminated, however, DA-3/sec cells form progressively growing tumors in all cases. These results suggest a crucial need for both CD4⁺ and CD8⁺ T lymphocytes in the rejection of DA-3/sec. Indeed, DA-3/sec tumors in T cell-depleted mice begin to appear at the time that an adaptive CTL response normally will initiate, further suggesting an important role for T lymphocytes in DA-3/sec elimination.

Ag-specific T cell responses are a favorable marker for tumor rejection and can lead to regression and elimination of tumors in vivo (28). Because T lymphocytes are necessary for the rejection of DA-3/sec, we assessed the ability of mice challenged with DA-3/sec to generate CTL. We observed significant killing of DA-3/sec target cells, but not DA-3, DA-3/neo, or DA-3/TM cells, suggesting that DA-3/sec-specific CTL are being generated in response to tumor challenge. In vitro cytotoxicity assays have shown that BALB/c mice challenged with the parental DA-3, DA-3/TM, or DA-3/neo tumor cells do not generate cytotoxicity against their respective targets (data not shown), indicating that the DA-3 tumor background does not elicit a cytotoxic T cell response. Indeed, DA-3/sec-injected mice are not protected against a challenge from either DA-3 parental tumor cells or cells expressing the tandem repeat, demonstrating specificity toward Ag expressed by DA-3/sec cells (data not shown).

As Fig. 2,A demonstrates, DA-3/sec cells form tumors in nude mice, albeit with a significant delay in growth compared with either DA-3/neo or DA-3/TM tumor. To determine whether innate components protect against DA-3/sec tumor formation, mice deficient in NK, NKT, macrophages, or IFN-γ have been used. The results of these experiments demonstrate that a lack of each of the aforementioned components renders the host sensitive to initial DA-3/sec tumor formation, but continued growth of the DA-3/sec tumors, in all cases, is kept small and transient. The innate system provides several important mechanisms for controlling tumor growth, including direct cytotoxic activity, Ag presentation, and secretion of cytokines and chemokines that, in turn, recruit and activate other immune components (29). Mechanisms the innate immune cells use to control initial DA-3/sec formation include IFN-γ (Fig. 3 D). Indeed, histologic sections of mice implanted with DA-3/sec cells reveal very little tumor present at the injection site by day 3, in contrast to sections of DA-3, DA-3/TM, and DA-3/neo tumors (data not shown), suggesting that the majority of DA-3/sec cells are killed early after implantation.

During the depletion experiments, the appearance of DA-3/sec tumors varied slightly depending on which cell type was absent (Table I). Depletion of NKT cells led to the earliest formation of DA-3/sec tumors, followed by macrophages, IFN-γ, and, finally, NK cells. Our data demonstrate that DA-3/sec tumors appear earliest in NKT-deficient animals, suggesting that these cells may be the first to be stimulated by the tumor itself. Recent work has shown that NKT cells can activate NK and macrophages by secretion of cytokines such as IFN-γ (30, 31). It has yet to be determined whether the NKT cells in our system have a direct cytolytic effect on DA-3/sec cells, enhancing Ag presentation, or both. The role that these cells play in DA-3/sec rejection does not seem to be essential, because progressive tumor formation does not occur in CD1d^−/− mice, yet they do appear to improve CTL function (Fig. 5 B). Indeed, NKT cells have recently been shown to act as helper cells through dendritic cell-dependent CTL priming (22).

Recruitment and activation of immune cells is of primary importance in the recognition and elimination of tumor. We addressed whether DA-3/sec preferentially recruited host cells compared with DA-3/TM in vivo. DA-3/sec cells or tissue culture supernatants injected i.p. recruited significantly more monocytes/macrophages than DA-3/TM cells or supernatants (Table II). Interestingly, the exudates from DA-3/TM-injected animals contained significantly more polymorphonucleocytes (GR-1^high) than DA-3/sec exudates. We have also demonstrated that DA-3/sec-elicited peritoneal cells can stimulate naive and, to a greater extent, DA-3/sec-primed T cells to produce IFN-γ (Table III). These results suggest that the recruitment of accessory cells may play an important role in attenuating progressive DA-3/sec tumor formation through the activation of tumor-specific T cells.

The above-described assays also point to a secreted factor(s) responsible for monocyte/macrophage recruitment. During the RNA analysis of the tumor cells, we did observe a higher expression level of the neutrophil chemokine LIX in DA-3 and DA-3/TM cells compared with DA-3/sec. However, the significance of this finding has yet to be determined. The RNA analysis also led to the discovery that DA-3/sec cells have a high level of mRNA for the chemokine CCL2. RT-PCR and ELISA results confirmed that the MUC1/sec-expressing cells greatly overexpress CCL2 compared with other cell types. The mechanism of CCL2 up-regulation in MUC1/sec-producing cells is currently being evaluated. Unpublished observations from our laboratory, however, suggest that MUC1/sec controls CCL2 production, because the addition of anti-MUC1/sec Ab to DA-3/sec cells decreased CCL2 levels found in the supernatant by 25% (data not shown). The CC chemokine CCL2 is secreted by a broad range of cell types in response to proinflammatory cytokines and induces chemotaxis and activation of monocytes, NK cells, and T lymphocytes (32, 33). Tumor cells have also been shown to secrete CCL2 and that may or may not be beneficial to tumor cell survival, depending on several factors, including the amount of CCL2 produced and the timing and site of tumor formation (34, 35, 36). Indeed, in one model system high levels of CCL2 at the start of challenge led to tumor regression in melanoma cells, whereas lower levels led to tumor formation (34). A lower, less sustained secretion of CCL2 may, in fact, benefit the tumor by failing to recruit large numbers of inflammatory cells and also inducing a proangiogenic state surrounding the tumor, providing it the necessary nutrients and gas exchange (37). Therefore, the failure of DA-3/sec tumors to grow in wild-type mice may be partially attributable to their ability to secrete high levels of CCL2, leading to recruitment and activation of immune cells, such as monocytes and T lymphocytes.

The direct contribution of MUC1/sec to tumor regression is still currently unknown. Recently, the expression patterns of MUC1 and its isoforms have been examined in ovarian (38) and cervical (39) tumors from patients. These data show that most isoforms of MUC1 can be detected at the RNA level in transformed cells, but there does not seem to be a strong correlation between tumor grade and any particular MUC1 isoform. However, the ovarian carcinoma study also examined the expression of MUC1 isoforms in malignant and benign tumors and demonstrated that MUC1/sec was expressed only in patients with benign tumors (38). In contrast, eight other MUC1 variants could be detected in malignant ovarian carcinomas. The authors conclude that loss of MUC1/sec expression may lead to a malignant phenotype. MUC1/sec has also been shown to be expressed by several primary cell lines, including uterine, mammary, and prostate epithelial cells, and appears to be down-regulated after stress induced by serum starvation (40). The function of MUC1/sec in primary cells has yet to be determined, but it can act as a ligand for the signaling receptor, MUC1/Y (11); however, the genes induced by this receptor-ligand engagement are unknown. In our system the introduction of MUC1/sec into DA-3 mammary adenocarcinoma cells resulted in differential gene expression compared with parental DA-3 cells and DA-3 cells expressing the transmembrane form of MUC1. Current studies have begun to address the mechanisms of MUC1/sec gene induction.

Together our results demonstrate that cells expressing MUC1/sec fail to grow in vivo and that T lymphocytes mediate control of progressive DA-3/sec tumor growth. We also show that the MUC1/sec gene, when transfected into cells, induces gene expression, including large amounts of CCL2 that may contribute to tumor regression.

We are particularly grateful for the excellent technical assistance of Mantley Dorsey, Jr.