Annals of Surgical Oncology Sign the Guestbook
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
10.1245/ASO.2004.05.027
Annals of Surgical Oncology 11:328-339 (2004)
© 2004 Society of Surgical Oncology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pockaj, B. A.
Right arrow Articles by Mukherjee, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pockaj, B. A.
Right arrow Articles by Mukherjee, P.
Related Collections
Right arrow Other Breast

ORIGINAL ARTICLES

Reduced T-Cell and Dendritic Cell Function Is Related to Cyclooxygenase-2 Overexpression and Prostaglandin E2 Secretion in Patients With Breast Cancer

Barbara A. Pockaj, MD, Gargi D. Basu, PhD, Latha B. Pathangey, MS, Richard J. Gray, MD, Jose L. Hernandez, BA, Sandra J. Gendler, PhD and Pinku Mukherjee, PhD

From the Departments of Surgery (BAP, RJG), Biochemistry and Molecular Biology (GDB, LBP, JLH, SJG, PM), and Biostatistics (JLH), Mayo Clinic College of Medicine, Scottsdale, Arizona.

Correspondence: Address correspondence and reprint requests to: Pinku Mukherjee, PhD, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 13400 E. Shea Blvd., Scottsdale, AZ 85259; Fax: 480-301-7017; E-mail: mukherjee.pinku{at}mayo.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: In several neoplastic diseases, including breast cancer, immunosuppression correlates with disease stage, progression, and outcome. Thus, thorough analysis of immune parameters in breast cancer patients may be beneficial in designing effective anticancer immune-based therapies.

Methods: We investigated dendritic cell and T-cell function in breast cancer patients at various stages of the disease and in age-matched controls. We also evaluated cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) levels within the tumor milieu and in the circulation.

Results: T cells from cancer patients showed decreased proliferation in response to CD3 antibody stimulation. Analysis of T-cell helper type 1 and 2 cytokines revealed reduced levels of interferon-{gamma}, tumor necrosis factor-{alpha}, interleukin (IL)-12, and IL-2 and increased levels of IL-10 and IL-4. Dendritic cells from these patients showed significantly reduced expression of co-stimulatory molecules (B7 and CD40) and demonstrated reduced phagocytic ability, reduced antigen presentation to T cells, and reduced ability to mature in response to lipopolysaccharide. Data revealed increased synthesis of PGE2, an immune suppressor, along with increased expression of COX-2, a key regulator of PGE2 synthesis.

Conclusions: COX-2–induced PGE2 may contribute to immunosuppression and may directly block antitumor immunity while promoting tumor growth, providing us with the rationale for using COX-2 inhibition combined with immunotherapy.

Key Words: Cyclooxygenase-2 • Prostaglandin E2 • Dendritic cells • T cells • Breast cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The defective function of the host’s immune system is one of the major mechanisms by which tumors evade immune surveillance. T-cell anergy is thought to be an early event in tumor progression and may precede the generalized immunosuppression that is observed in cancer patients.1 Multiple mechanisms of tumor-specific evasion have been demonstrated, including impaired HLA expression, modulation of surface antigens, lack of co-stimulatory molecules on dendritic cells (DCs) leading to impaired antigen presentation to T cells, impaired T-cell receptor (TCR) signal transduction through the TCR-{zeta} chain, and elaboration of immune-suppressive cytokines by tumor cells and T-regulatory cells such as interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß).2

Functional impairment of T cells has been well documented in patients with cancer.3 This is true of both circulating and infiltrating lymphocytes in which there is reduced activation of the TCR and impaired production of interferon-{gamma} (IFN-{gamma}), IL-2, and tumor necrosis factor-{alpha} (TNF-{alpha}) in response to CD3 monoclonal antibodies.4 Cytokine profiles of cancer patients demonstrate an abnormal balance between T-cell helper type 1 (Th1) and type 2 (Th2) cytokines, favoring a Th2 response.5 Circulating and tumor-infiltrating DCs have also been shown to be functionally impaired. In metastatic melanoma patients, tumor-infiltrating DCs express low levels of co-stimulatory molecules (CD80 and CD86) and therefore are unable to activate T cells, whereas DCs isolated from breast cancer patients demonstrate a significantly reduced ability to stimulate allogeneic and antigen-specific T-cell responses.6,7 In certain cancers, DCs derived from peripheral blood are lower in absolute number as compared with those in individuals without cancer and are predominantly immature in phenotype.8

Cyclooxygenase-2 (COX-2) is overexpressed in a variety of cancers, including breast cancer.9–11 COX-2 is an enzyme that converts arachidonic acid to prostaglandin H2, which is further metabolized to other prostaglandins, including prostaglandin E2 (PGE2).12 COX-2 expression is rapidly induced secondary to a number of factors, including growth factors, tumor promoters, and hormones.13 Transgenic mouse models demonstrated that overexpression of COX-2 leads to the development of mammary tumors.14 Overexpression of COX-2 is also known to inhibit apoptosis15 and promote angiogenesis.16 This overexpression of COX-2 can lead to increased production of prostaglandins such as PGE2, which has multiple downstream effects. PGE2 is known to transactivate the epidermal growth factor receptor, which triggers mitogenic signaling in epithelial cells and induces cancer cell proliferation.17 PGE2 also causes immunosuppression in vitro18 and can induce immunosuppression in vivo, enhancing tumor growth in animal models.19,20 In this study, we tested the hypothesis that the COX-2–induced PGE2 overexpression may correlate with the global immunosuppression observed in breast cancer patients.

Because T cells and DCs are pivotal in the development of antitumor immunity and are susceptible to tumor-mediated immune suppression, we investigated DC and T-cell function from 25 breast cancer patients at various stages of the disease and compared the data with those of 19 healthy age-matched controls. Although several studies have described the functional impairment of T cells and DCs in breast cancer patients, the studies have not evaluated both T-cell and DC function from the same breast cancer patients. Moreover, the mechanisms driving the functional impairment still remain elusive. The goal of our study was to evaluate the immune status of patients presenting with the diagnosis of breast cancer and to evaluate the immune-modulating factors within the tumor milieu that may account for the functional impairment of immune effector cells. This is the first study to describe a thorough analysis of both T-cell and DC function in patients with newly diagnosed breast cancer. Impaired functionality of T cells and DCs correlated with COX-2 and PGE2 overexpression. These studies are of critical importance for designing novel immunotherapeutic strategies for breast cancer and for selecting the patients who may most benefit from such therapies.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Characteristics
This research study was approved by the Mayo Clinic Institutional Review Board. Patients who presented to the Mayo Clinic Scottsdale Breast Clinic for initial treatment of disease were eligible for the protocol. The patients signed informed consent for peripheral blood and tumor samples. Informed consent for peripheral blood samples was also obtained from healthy, age-matched volunteers. Twenty-five patients with breast cancer and 19 healthy controls were studied. Patients and healthy donors who were chronic users of COX-2 inhibitors were excluded from the analysis. Staging of the patients was conducted by using the American Joint Committee on Cancer protocol. Breast cancer patient demographics and tumor characteristics are listed in Table 1. All patients were postmenopausal, with a mean age of 69 years. Controls were all postmenopausal, with a mean age of 60 years. Infiltrating ductal adenocarcinoma was the most common tumor subtype (64%). The mean size of the tumors was 2.3 cm, although 64% of lesions were <=2 cm. Only 20% of patients had lymph node metastases, and most patients presented with either stage I or stage II disease. In most cases, blood was drawn on the day of surgery, before resection of the tumor. In some cases, blood was drawn few days before surgery.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Patient demographics and tumor characteristics
 
The overall scheme for the study design is shown in Fig. 1. Whole blood was obtained from the study subjects, and the peripheral blood mononuclear cells (PBMCs) were separated by using a Ficoll-Paque density gradient centrifugation. Sera from these patients were also collected and stored in a -80°C freezer. The PBMCs were used for isolation of T cells and DCs, and the serum was used for cytokine/chemokine and PGE2 evaluation. Surgically resected tumor tissues and lymph node metastases were used to make tissue lysate for COX-2 and PGE2 evaluation.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1. Schematic representation of the study design. COX-2, cyclooxygenase-2; TCR, T-cell receptor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; TNF, tumor necrosis factor; AIM-V, serum free human lymphocyte culture media.

 
T-Cell Isolation
T cells were isolated from the PBMCs of patients as described in the scheme (Fig. 1). Briefly, mononuclear cells were obtained by centrifugation of peripheral blood over a Ficoll-Paque gradient (Amersham Biosciences, Uppsala, Sweden). Mononuclear cells were incubated for 2 hours at 37°C, and nonadherent lymphocytes were removed and used as T cells and adherent cells were used for generation of DCs. The processing of the blood was performed under the same conditions, and all assays were performed on freshly isolated buffy-coat cells.

T-Cell Proliferation Assay
The nonadherent lymphocyte population (1 x 106/mL) was subjected to in vitro stimulation with various concentrations of purified plate-bound CD3 antibody (BD Pharmingen, San Diego, CA). Cells were incubated for 4 days with CD3 antibody, and 3H-thymidine was added 24 hours before collection. After excess thymidine was washed off, cells were lysed with 5% Triton X-100 (Fischer Scientific, Pittsburgh, PA), and incorporated thymidine was evaluated with the Topcount micro scintillation counter (Packard Biosciences, Shelton, CT). Evaluation of T-cell proliferation was also performed with varying concentrations of tumor lysates (12–200 µg/mL) and purified PGE2 (Cayman Pharmaceuticals, Ann Arbor, MI). All assays were performed in triplicate. Control lymphocytes were included in every assay to control for interassay variation.

Analysis of Intracellular Cytokines
Intracellular cytokine levels were evaluated by two-color flow cytometric analysis after TCR ligation. Intracellular cytokines were determined post brefeldin A (BD Pharmingen) treatment of lymphocytes according to the manufacturer’s recommendations (4 µL/1.2 x 107 cells per 6 mL for 3 hours at 37°C before staining). This treatment stops the release of cytokines in the culture media, and the cytokines accumulate within the cells. Cells were then stained for surface markers for T cells (CD3) or DCs (HLA-DR) at 4°C for 15 minutes, followed by washing excess stain and permeabilizing cells with Pharmingen permeabilization solution (containing saponin) for 30 minutes at 4°C. Cells were then stained for intracellular IL-2, IL-12, IFN-{gamma}, IL-4, IL-10, and TNF-{alpha} for 30 minutes at 4°C. Cells were analyzed with the Becton Dickinson FACScan, and data were analyzed with the CellQuest program. All antibodies were purchased from BD Pharmingen.

Serum Analysis of Cytokines and Chemokines
A cytokine/chemokine array kit (Ray Biotech Inc., Norcross, GA) was used to detect a panel of 22 secreted cytokines and chemokines in the serum from healthy patients and those with breast cancer. The manufacturer’s recommended protocol was used.

DC Isolation and Maturation
DCs were generated from a CD14+ monocyte population isolated from PBMCs. Briefly, mononuclear cells were obtained by centrifugation of the peripheral blood over a Ficoll-Paque gradient. Mononuclear cells were incubated for 2 hours at 37°C, and nonadherent cells were removed. Adherent cells were incubated with granulocyte-macrophage colony-stimulating factor (5 ng/mL; PeproTech, Rocky Hill, NJ) and IL-4 (5 ng/mL; PeproTech) for 4 to 5 days. Cells were collected, counted, and phenotyped for immature DCs and were further cultured for one additional day with granulocyte-macrophage colony-stimulating factor (5 ng/mL), IL-4 (5 ng/mL), and lipopolysaccharide (LPS, 100 ng/mL; Sigma Pharmaceuticals, St. Louis, MO). Cells were collected on day 6 as mature DCs.

DC Phenotype
Control and breast cancer patient DCs were analyzed by two-color flow cytometric analysis. Cell-surface expression of several markers was evaluated: CD80 (B7.1), CD86 (B7.2), CD40, HLA-DR, HLA-ABC, CD1a, and CD14. All antibodies were purchased from BD Pharmingen. Stained cells were analyzed with the CellQuest program on a Becton Dickinson FACScan.

DC Function
Mixed Lymphocyte Reaction Assay
Control and patient-derived DCs were assayed for their ability to stimulate allogeneic T cells in a mixed lymphocyte reaction (MLR). T cells (1 x 105) from healthy donors were incubated with irradiated DCs (3000 rads; 1 x 104 cells) from allogeneic breast cancer patients for 5 days, and 3H-thymidine was added 24 hours before the cells were collected. After the excess thymidine was washed off, cells were lysed with 5% Triton X-100, and incorporated thymidine was evaluated by using the Topcount micro scintillation counter.

Phagocytosis Ability
Immature and LPS-matured DCs from healthy donors and breast cancer patients were incubated with fluorescein isothiocyanate–conjugated dextran beads (molecular weight, 40,000; Molecular Probes Inc., Eugene, OR) at 1 mg/1 x 106 cells for 30 minutes at 37°C. Dextran beads were used as the exogenous antigen source. Because the beads were conjugated to fluorescein isothiocyanate, uptake of dextran beads by DCs was analyzed by flow cytometry, and mean fluorescence intensity was calculated.

Breast Tumor Cell Lysates
Tissue lysates were prepared within 1 hour after surgery by homogenizing the tumor tissue in lysate buffer containing 20 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, .15 M of NaCl, and 1% Triton X-100 supplemented with phosphatase-inhibitor cocktail mix (1/100 dilution; Sigma Pharmaceuticals) and complete protease inhibitors (Roche Pharmaceuticals, Indianapolis, IN). Lysates were stored in a -80°C freezer for further use.

Expression of COX-2 Protein in Tumor Lysate
Protein concentrations of the lysates were determined with the Pierce BCA protein assay kit (Pierce, Rockford, IL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 12% resolving gel; 100 µg of protein was loaded per lane. Gels were immunoblotted and probed for COX-2 with specific COX-2 monoclonal antibody (goat polyclonal antibody, clone C20; Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a 1/200 dilution.

PGE2 in Serum and Tumor Lysate
Levels in the lysates were determined with a specific enzyme-linked immunosorbent assay (ELISA) kit for PGE2, and levels in serum were determined by using the PGE2 metabolite ELISA kit (Cayman Pharmaceuticals). The manufacturer’s recommended protocols were followed.

Statistical Analysis
Statistical significance was assessed by using pairwise comparisons with the Tukey-Kramer adjustment for multiple comparisons. The margins of error for the comparisons were obtained by calculating the 95% confidence intervals for the differences between group proportions.

Immune function parameters were compared between a set of clinical indicators. The clinical parameters examined were stage, lymph node status, estrogen receptor status, tumor size (<2 vs. 2 cm), grade, presence of angiolymphatic invasion, multifocality, and previous breast cancer. Because of the nonnormality of the immune function data and the small sample size of the cohort, the exact Wilcoxon statistic was used for assessing significant differences between groups. All error bars in the figures represent the standard deviation of the mean.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The proliferative ability of T cells isolated from breast cancer patients just before surgical tumor resection was examined. T cells were stimulated by various concentrations of plate-bound CD3 antibody, and T-cell proliferation was measured by 3H-thymidine uptake. T-cell proliferation was significantly reduced in cancer patients compared with controls (P < .001 at 1 µg and .5 µg/mL of CD3 antibody; Fig. 2A). The raw counts per minute for all patients (n = 25) and controls (n = 19) are presented in Fig. 2B for a 1 µg/mL CD3 antibody concentration. Figure 2B clarifies the number of patients who were immunosuppressed. Sixty percent (16 of 25) of the breast cancer patients had lower than 50,000 counts per minute, which is suggestive of impaired T-cell proliferation in response to TCR ligation. All controls had values greater than this value. Additional analysis of Th1/Th2 cytokines in activated cells revealed reduced intracellular levels of the immunostimulatory Th1 cytokines IFN-{gamma} (P < .001), TNF-{alpha} (P < .001), IL-12 (P < .001), and IL-2 (P < .001) and increased levels of the Th2 cytokines IL-4 (P = .1) and IL-10 (P = .01; Fig. 3).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2. T cells from breast cancer patients have markedly reduced proliferation in response to specific T-cell receptor ligation. (A) T-cell proliferation in response to plate-bound CD3 antibody comparing breast cancer patients (n = 25) and controls (n = 19). The amount of 3H-thymidine uptake directly corresponds to the proliferative capacity of T cells. Error bars represent standard deviation of the mean. All assays were performed in triplicate with interassay controls. (B) Scatter plot of 3H-thymidine uptake (in counts per minute; CPM) of T cells from individual patients (n = 25) and healthy donors (n = 19) in response to 1 µg/mL of plate-bound CD3 antibody.

 


View larger version (25K):
[in this window]
[in a new window]
  		FIG. 3. Reduced T-helper type 1 (Th1) cytokines and increased T-helper type 2 (Th2) cytokines in the peripheral blood of patients with breast cancer. Intracellular cytokine levels were compared by flow cytometry. Interferon (IFN)-{gamma}, tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-12, IL-2, IL-10, and IL-4 levels were compared between breast cancer patients (solid bars; n = 9) and controls (open bars; n = 7). Error bars represent standard deviation of the mean.

 
DCs from patients with breast cancer expressed reduced levels of co-stimulatory molecules such as CD80 (B7.1), CD86 (B7.2), and CD40 on maturation with LPS (Fig. 4). Similar results were obtained with TNF-{alpha}–induced maturation. Other markers used to determine DC phenotype included HLA-DR, HLA-ABC, CD1a, and CD14 (data not shown). No differences between cancer patients and controls were observed in these markers. The reductions in expression of both CD80 and CD86 were statistically significant (P < .001). This low expression of co-stimulatory molecules is an indication that these DCs remain immature. It has been suggested (1) that immature DCs have a reduced ability for stimulating T cells and therefore may contribute to tumor-induced T-cell tolerance rather than immunity and (2) that mature DCs are essential for presenting tumor antigens and activating T cells to become cytolytic against tumor cells.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4. Dendritic cells (DCs) from patients with breast cancer expressed reduced levels of co-stimulatory molecules. Flow cytometric analysis is shown of the surface expression of the co-stimulatory molecules CD80 (B7.1), CD86 (B7.2), and CD40 on lipopolysaccharide-matured DCs of breast cancer patients (solid bars; n = 25) versus controls (open bars; n = 19). Similar results were obtained with tumor necrosis factor-{alpha}–matured DCs (data not shown). Error bars represent standard deviation of the mean.

 
We therefore tested whether DCs from cancer patients had reduced antigen presentation. Data indicate that the function of DCs was significantly reduced. DCs from breast cancer patients demonstrated a significantly reduced ability to present antigens to allogeneic normal T cells in an MLR (P < .001; Fig. 5A). The MLR results support the previous findings shown in Fig. 4, in which the same DCs expressed low levels of co-stimulatory molecules and therefore failed to fully mature. Furthermore, the immature DCs from breast cancer patients demonstrated a significantly reduced ability to phagocytose exogenous antigens in vitro (P < .001; Fig. 5B and C). They also demonstrated a maturation defect when stimulated with LPS treatment (Fig. 5C). Figure 5B shows a representative histogram from 1 patient, and Fig. 5C shows a bar graph of average values from the 24 patients and 19 controls. Immature DCs are known to be strong phagocytes but weak antigen presenters, whereas mature DCs are weak phagocytes and strong antigen presenters.21 We found that immature DCs from healthy donors showed significantly higher (P < .001) fluorescence intensity (mean intensity of 1436), thus demonstrating good phagocytic ability, and on maturation with LPS, their phagocytic activity was reduced (mean intensity of 291; Fig. 5C). In comparison, the immature DCs from the breast cancer patients had significantly lower fluorescence intensity (mean intensity of 337), thus indicating poor phagocytosis; the florescence intensity of these DCs did not decrease with LPS treatment and remained at mean intensity of 207, once again indicating impaired maturation (Fig. 5C). Similar results were obtained when TNF-{alpha} instead of LPS was used to mature DCs (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 5. Dendritic cells (DCs) from breast cancer patients demonstrate a significantly reduced ability to present antigens to allogeneic normal T cells and demonstrate reduced phagocytosis of exogenous antigen. (A) Allogeneic antigen presentation to normal T cells by DCs of breast cancer patients (n = 25) versus controls (n = 19) in a mixed lymphocyte reaction. The amount of 3H-thymidine uptake directly corresponded to the proliferative capacity of T cells. (B) Representative histogram of immature and lipopolysaccharide-matured dendritic cell phagocytic ability of a cancer patient versus healthy donor (the numbers on the right corner are the mean fluorescence intensity). (C) Dendritic cell phagocytic ability (mean fluorescence intensity) in breast cancer patients (solid bars; n = 25) versus normal controls (open bars; n = 19) in the mature and immature state. In (B) and (C), the mean fluorescence intensity was used as a measure for the amount of fluorescein isothiocyanate–conjugated dextran beads engulfed by the DCs. Error bars represent standard deviation of the mean. CPM, counts per minute; FITC, fluorescein isothiocyanate.

 
Tumor cells secrete factors that are known to induce immunosuppression and promote tumor cell proliferation. Prostaglandins, especially PGE2, are such factors expressed within the tumor microenvironment and secreted in the serum. Because COX-2 is the key regulator of prostaglandin synthesis, we evaluated the COX-2 protein expression on Western blots of adjacent normal tissue lysates, tumor tissue lysates, and lysates from lymph nodes that contained metastases. The COX-2 protein was overexpressed in both the tumor and lymph node metastases compared with normal tissue. The highest expression was observed in lymph node metastases (Fig. 6A). Next, we evaluated the amount of PGE2 in the serum of breast cancer patients and healthy donors by specific PGE2 metabolite ELISA. Because PGE2 is rapidly converted in the serum by 15-OH prostaglandin dehydrogenase to its 13,14-dihydro-15-keto metabolite, direct measurement of intact PGE2 is not possible in sera or plasma. Thus, PGE2 metabolite measurement is necessary to provide a reliable estimate of actual PGE2 production.22,23 However, in the tumor tissue lysates, PGE2 levels can be evaluated directly by using a specific ELISA for PGE2, because these cell types do not contain the enzymes required for metabolism of PGE2, thus keeping the PGE2 levels stable. Breast cancer patients had significantly increased levels of PGE2 metabolite levels in their sera as compared with controls (P < .001; Fig. 6C). Similar to COX-2 expression, we observed high levels of PGE2 in the tumor cell lysates (P = .042) and lymph node metastases (P = .017) as compared with normal adjacent breast tissue (Fig. 6B).



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 6. Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are overexpressed in the tumor tissue and serum of patients with breast cancer. (A) COX-2 protein levels in tissue lysates (100 µg) from adjacent normal breast tissue, breast tumors, and lymph node metastases were analyzed by Western blot analysis. Brackets indicate tissues from a single patient. Six patient samples were analyzed. (B) PGE2 levels in tissue lysates from adjacent normal breast tissue, breast tumors, and lymph node metastases were determined by specific enzyme-linked immunosorbent assay (ELISA). (C) Serum PGE2 metabolite levels in breast cancer patients (solid bars) versus controls (open bars) were analyzed by specific PGE2 metabolite ELISA. Error bars represent standard deviation of the mean. N, normal adjacent tissue; T, tumor; LN, lymph node metastasis; IB, immunonoblot.

 
Because tumor lysates contained COX-2, PGE2, and, presumably, other immunosuppressive factors, we determined whether these lysates could directly block the T-cell signal transduction and activation that lead to T-cell proliferation. We evaluated the effect of the tumor lysates on the proliferation of T cells from the healthy donors. We used purified PGE2 as a standard and compared it with the inhibition observed with the tumor lysate and lysate derived from adjacent normal tissue. There was direct inhibition of normal T-cell proliferation in response to CD3 antibody by the tumor lysates (n = 3) and lysates derived from lymph node metastasis (n = 2) at a 25 µg/mL concentration (Fig. 7). As expected, inhibition was also observed with purified PGE2 at the same concentration (Fig. 7). Little inhibition of T-cell proliferation was observed with lysates derived from adjacent normal tissue, which did not express high levels of PGE2 by ELISA (Fig. 6B). The inhibition observed with tumor and lymph node metastasis lysate was higher than with purified PGE2, suggesting the presence of other T cell–inhibitory agents in the tumor lysate. Because normal lymph nodes express some PGE2, normal adjacent tissue may not be the ideal control for lymph node metastasis lysate. Thus, we evaluated normal lymph node lysates from mice (because normal human lymph nodes were not available) and determined that the PGE2 levels in normal lymph nodes were minimal and were not sufficient to cause inhibition of mouse T-cell proliferation in response to TCR ligation (data not shown). Moreover, in humans, it has been well established that normal T cells that have not undergone any antigenic challenge or environmental insult generate very little PGE2. In vitro studies have proven that normal T cells have to be stimulated with mitogen to express PGE2.24,25 Thus, we are confident that the inhibition of T-cell proliferation that we observed with lysates from lymph node metastasis was due to high levels of PGE2 (Fig. 6B) and other unknown immunosuppressive factors.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 7. Direct inhibition of normal T-cell proliferation by factors present in the tumor lysate. T-cell proliferation was determined in response to plate-bound CD3 antibody in the presence or absence of lysate derived from the primary tumor, lysate derived from lymph node (LN) metastasis, lysate derived from adjacent normal tissue, or purified prostaglandin E2 (PGE2) at varying concentrations (0 –200 µg/mL). T cells were generated from healthy donors (n = 4). The amount of 3H-thymidine uptake directly corresponds to the proliferative capacity of T cells. Tissue lysis buffer and lysate from adjacent normal tissue were used as negative controls, and purified PGE2 was used as the positive control. This assay was repeated three times, with similar results. CPM, counts per minute.

 
We evaluated our data to see whether the patient’s clinical variables correlated with immune function parameters. The clinical parameters examined were stage, lymph node status, estrogen receptor status, tumor size (<=2 vs. 2 cm), grade, presence of angiolymphatic invasion, multifocality, and previous breast cancer. Immune function variables were compared between a set of clinical indicators. Because of the nonnormality of the immune function data and the small sample size of the cohort, the exact Wilcoxon statistic was used in assessing significant differences between groups. However, no significant correlation was reached except for impaired DC phagocytosis and antigen presentation in patients with lymph node–positive disease versus those who were lymph node negative (P = .025). The inability to elucidate any differences in any other parameters and clinical factors is due to small sample size.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is evidence that tumor-specific antigens are present on cancer cells that could function as potential targets for the immune system. Unfortunately, cancer patients do not mount an effective immune response against them, indicating that the immune cells are tolerant to the tumor-specific antigens. Breaking this tolerance is one of the major goals of immunotherapy for cancer. Tumors also exhibit multiple immunosuppressive strategies, such as downregulation of major histocompatibility complex class I molecules, lack of co-stimulatory molecules on DCs, and secretion of immunosuppressive cytokines, as well as production of high levels of COX-2 and PGE2. We first examined the immune status of patients recently diagnosed with breast cancer and then evaluated the effect of COX-2 overexpression by the tumor cells and subsequent synthesis of PGE2 on the tumor’s ability to evade immune surveillance.

Overall we found significant functional impairment in the T cells of patients who were diagnosed with breast cancer. T cells isolated from breast cancer patients before surgical removal of the tumor demonstrated a marked reduction in their proliferation response to CD3 antibodies (Fig. 2), suggesting a defect in activation of the TCR-mediated signal transduction pathways.26 These described defects include reduced TCR-{zeta} chain expression, a defect in transcription factors such as nuclear factor-{kappa}B, upregulation of cyclin-dependent kinase inhibitor p27kip1, and hydrogen peroxide production by activated granulocytes.26–30 A likely consequence of this ineffective T-cell signaling is impaired cytokine production by the T cells.5,31 Nieland et al.31 found that reduced cytokine expression was found in patients with early breast cancer with a normal TCR-{zeta} chain, suggesting involvement of other mechanisms in causing impaired cytokine production.

Th1 cytokines promote the development of cell-mediated antitumor responses.32 However, Th2 cytokines are necessary for humoral immunity. Patients with carcinoma have a predominance of Th2 cytokines in the peripheral blood.5 We found a similar increase in Th2 cytokines (IL-10 and IL-4) in our breast cancer cohort when compared with controls and found reduced Th1 cytokines (IFN-{gamma}, IL-2, and IL-12; Fig. 3). A shift to a Th2 response has been correlated with increasing stage in patients with renal cell carcinoma.33 Preliminary analysis of serum chemokine and cytokine levels by using a cytokine array system revealed a correlation between increasing levels of certain cytokines and chemokines with advanced-stage breast cancer (Fig. 8). RANTES (regulated on activation, normal T cells expressed and secreted), monokine induced by IFN-{gamma}, monocyte chemoattractant protein-1, IL-8, and IL-10 levels (proteins that favor a Th2 response) were higher in the sera from patients with high-grade tumor and lymph node metastases as compared with patients with low-grade tumor and no lymph node metastases. Monocyte chemoattractant protein-1 is implicated in tumor cell migration and invasion and in multidrug resistance.34 Similarly, monokine induced by IFN-{gamma} and RANTES favor inflammation and tumor cell proliferation and invasion.35,36 IL-8 is implicated in increased angiogenesis and multidrug resistance,34 and IL-10 is known to cause T-cell anergy.37 TGF-ß1 is known to downregulate both cytotoxic T lymphocyte and T-helper signal transduction and function.38



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 8. Serum analysis of the chemokine/cytokine array revealed a correlation between increasing levels of certain cytokines and chemokines and advanced-stage breast cancer. Expression of a panel of 22 secreted cytokines and chemokines was detected in the serum of healthy and breast cancer patients by using the Ray Biotec cytokine array kit. Sera are shown from one control, one patient with a grade 1 invasive breast cancer without lymph node metastases, and one patient with a grade 3 invasive breast cancer with lymph node metastases. Similar results were observed with the other five breast cancer patients tested. The boxes on the blots and table demonstrate the cytokines and chemokines that are either upregulated (+) or downregulated (-) compared with the normal serum. The open box represents the immunostimulatory cytokines, the gray box represents chemokines that favor aggressive tumor growth, and the black box represents immunosuppressive cytokines. The actual cytokine array map from Ray Biotec is also provided. IFN, interferon; TNF, tumor necrosis factor; MCP, monocyte chemoattractant protein; RANTES, regulated on activation, normal T cells expressed and secreted; MIG, monokine induced by interferon-{gamma}; IL, interleukin; TGF, transforming growth factor; GCSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GRO, growth-related oncogene.

 
These chemokines and cytokines are released by monocytes, macrophages, and lymphocytes that express the EP receptors for binding to PGE2.39 It is therefore plausible that the high levels of PGE2 observed within the tumor microenvironment and in the circulation may activate the tumor-infiltrating lymphocytes, monocytes, and macrophages via the EP receptors to release factors that favor tumor growth and invasion and suppress immune effector cells. These proteins were either absent or present in very low levels in the sera of controls. Conversely, serum levels of the immunostimulatory cytokines TNF-ß1 and IFN-{gamma} were higher in patients with low-grade/nonmetastatic tumors when compared with patients with high-grade/metastatic tumors, once again suggesting that advanced-stage patients are more likely to be immunosuppressed. The cytokine/chemokine array analysis was performed on six breast cancer patients and three healthy donors. Representative blots from two cancer patients and one healthy donor are shown in Fig. 8. The tumor milieu has been thought to play a significant role in the impairment of DCs. Release of such factors as IL-6, IL-10, macrophage colony-stimulating factor, vascular endothelial growth factor, and macrophage inflammatory protein-3{alpha} can prevent DC maturation and antigen-presenting functions. It is interesting to note that in our preliminary analysis of sera from four patients with breast cancer, we observed increased levels of vascular endothelial growth factor, macrophage colony-stimulating factor, IL-10, and macrophage inflammatory protein-3{alpha} (data not shown) as compared with controls. These results fit well with the concept that these increased chemokine levels favor a Th2 response that limits the DC allostimulatory capability.7,40–42 In one study, it was shown that surgical removal of the primary tumor resulted in a dramatic reduction in the proportion of immature DCs, although the levels never reached those of controls.8

Mature DCs are the most powerful antigen-presenting cells and thus initiate the immune response.21 The presence of immature DCs is thought to contribute to the induction of tolerance instead of immunity against the tumor antigens.43 Low expression of co-stimulatory molecules on circulating DCs is an indication of immaturity. Low levels of co-stimulatory molecules in peripheral and draining lymph node DCs of breast cancer patients and impaired allostimulatory ability have been demonstrated in patients with breast cancer.7,8 Our study confirms some of these findings as shown by the low expression of co-stimulatory molecules on circulating DCs from our breast cancer patients (Fig. 4). Our data further demonstrate the immaturity of DCs by the fact that DCs from cancer patients have a reduced ability to present antigens to allogeneic normal T cells in an MLR assay (Fig. 5A).

Heightened endocytic activity is characteristic of cytokine-derived DCs and their enhanced capacity to capture and process antigens.21 Our study demonstrated impaired phagocytosis by the immature DCs from breast cancer patients (Fig. 5B and C). Attempts to mature the DCs with LPS did not change their phagocytic ability, once again suggesting a defect in DC maturation.

The mechanisms that underlie the T-cell and DC anergy in cancer patients are unknown but probably involve multiple events. We evaluated whether overexpression of COX-2 and downstream PGE2 synthesis may be one of the mechanisms for immunosuppression. It is interesting to note that the COX-2 expression was high in primary tumors and was even more prominent in lymph node metastases (Fig. 6A). Because COX-2 was overexpressed, we evaluated the PGE2 levels in the serum of the breast cancer patients and controls along with tumor lysates. Increased levels of PGE2 were demonstrated in both the sera and tumor lysates of patients with cancer (Fig. 6B and C). PGE2 is an immune suppressor that targets both cytotoxic and helper T-cell functions. PGE2 is thought to suppress cell-mediated immune responses while enhancing humoral immune responses.44,45 PGE2 suppresses chemokine and cytokine production in humans, including IFN-{gamma}–, TNF-{alpha}–, IL-12–, and IL-1ß–mediated expression of chemokines. PGE2 upregulates expression of immunosuppressive cytokines, such as IL-10 and TGF-ß.45,46 This immunosuppressive effect of PGE2 was demonstrated by inhibition of normal T-cell proliferation to tumor lysates with high concentrations of PGE2 (Fig. 7).

The ability of mature DCs to act as potent antigen-presenting cells is related to their production of IL-12.47 DCs deficient of IL-12 generated in the presence of PGE2 promote a Th2 response.48 A recent study demonstrated that high concentrations of PGE2 caused decreased IL-12 production via increases in IL-10 production and, therefore, decreased DC function.18 This correlates well with our data, which clearly demonstrate increased PGE2 levels in the serum and within the tumor milieu and also show increased levels of intracellular IL-10 and decreased levels of IL-12 in the T cells of the breast cancer patient population.

Thus, tumor overexpression of COX-2 via the elaboration of PGE2 and other mechanisms could directly block the patient’s defense mechanism against cancer and promote breast cancer growth.49 We observed overexpression of COX-2 and PGE2 and impaired T-cell and DC function in breast cancer patients. If the immune system of breast cancer patients were persistently compromised, the success of immunotherapies would be limited unless the immune system could be appropriately stimulated. Many immunotherapies for cancer treatment have been partially successful in eliciting a cellular immune response; however, this response has been downregulated by tumor-derived immunosuppressive factors. If mediators of immune suppression, such as COX-2 and PGE2, can be reduced; if co-stimulation for cytotoxic T-lymphocyte effector functions can be provided with appropriate immune-based therapy to overcome the tolerizing effects of the tumor; and, most importantly, if tumor cell proliferation can be restricted, then immunotherapy can be very effective. This study, along with other studies in the literature, provides us with an immunological rationale for using COX-2 inhibition that would reduce the PGE2 levels and therefore reduce immunosuppression and tumor cell growth. COX-2 inhibition combined with immune-based therapy that would induce cytotoxic T-lymphocyte activity against tumor cells is a novel concept that needs further exploration in preclinical animal models and in clinical settings.


   ACKNOWLEDGMENTS
 
The acknowledgments are available online in the fulltext version at www.annalssurgicaloncology.org. They are not available in the PDF version.

Supported by the Susan G. Komen Breast Cancer Foundation, the Department of Defense Breast Cancer Research Program (grant DAMD17-01-1-0318), and The Mayo Comprehensive Cancer Center. The authors thank all the technicians in the radiation department for help with irradiation of cells; Heidi Apsey, RN, Marie Malikowski, RN, and Donna Passante, RN, for collection of specimens; Dr. Adams and all the technologists in the histopathology department for the tumor tissue samples; Jim Tarara for his help with flow cytometry; Marvin Ruona in visual communications for generating the figures; and Carol Williams for her assistance in the preparation and submission of the manuscript.


   FOOTNOTES
 
T-cell and dendritic cell function were found to be impaired in breast cancer patients. In addition, we found cyclooxygenase-2 (COX-2) overexpression and increased prostaglandin E2 (PGE2) levels in breast cancer. Data suggest a direct correlation between increased COX-2 and PGE2 expression and impaired immune cell function.

Received for publication May 27, 2003. Accepted for publication November 4, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Staveley-O’Carroll K, Sotomayor E, Montgomery J, et al. Induction of antigen-specific T cell anergy: an early event in the course of tumor progression. Proc Natl Acad Sci U S A 1998; 95: 1178–83.[Abstract/Free Full Text]
  2. Pardoll D. Does the immune system see tumors as foreign or self? Ann Rev Immunol 2003; 21: 807–39.[CrossRef][Medline]
  3. Finke J, Ferrone S, Frey A, Mufson A, Ochoa A. Where have all the T cells gone? Mechanisms of immune evasion by tumors. Immunol Today 1999; 20: 158–60.[CrossRef][Medline]
  4. Keilholz U, Weber J, Finke JH, et al. Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy. J Immunother 2002; 25: 97–138.[Medline]
  5. Goto S, Sato M, Kaneko R, Itoh M, Sato S, Takeuchi S. Analysis of Th1 and Th2 cytokine production by peripheral blood mononuclear cells as a parameter of immunological dysfunction in advanced cancer patients. Cancer Immunol Immunother 1999; 48: 435–42.[CrossRef][Medline]
  6. Enk AH, Jonuleit H, Saloga J, Knop J. Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma. Int J Cancer 1997; 73: 309–16.[CrossRef][Medline]
  7. Gabrilovich DI, Corak J, Ciernik IF, Kavanaugh D, Carbone DP. Decreased antigen presentation by dendritic cells in patients with breast cancer. Clin Cancer Res 1997; 3: 483–90.[Abstract]
  8. Almand B, Resser JR, Lindman B, et al. Clinical significance of defective dendritic cell differentiation in cancer. Clin Cancer Res 2000; 6: 1755–66.[Abstract/Free Full Text]
  9. Singh B, Lucci A. Role of cyclooxygenase-2 in breast cancer. J Surg Res 2002; 108: 173–9.[CrossRef][Medline]
  10. Singh-Ranger G, Mokbel K. The role of cyclooxygenase-2 (COX-2) in breast cancer, and implications of COX-2 inhibition. Eur J Surg Oncol 2002; 28: 729–37.[CrossRef][Medline]
  11. Half E, Tang XM, Gwyn K, Sahin A, Wathen K, Sinicrope FA. Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Res 2002; 62: 1676–81.[Abstract/Free Full Text]
  12. Herschman HR. Regulation of prostaglandin synthase-1 and prostaglandin synthase-2. Cancer Metastasis Rev 1994; 13: 241–56.[CrossRef][Medline]
  13. Herschman HR. Prostaglandin synthase 2. Biochim Biophys Acta 1996; 1299: 125–40.[Medline]
  14. Liu CH, Chang SH, Narko K, et al. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001; 276: 18563–9.[Abstract/Free Full Text]
  15. Munkarah AR, Morris R, Baumann P, et al. Effects of prostaglandin E(2) on proliferation and apoptosis of epithelial ovarian cancer cells. J Soc Gynecol Invest 2002; 9: 168–73.[Medline]
  16. Amano H, Hayashi I, Endo H, et al. Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth. J Exp Med 2003; 197: 221–32.[Abstract/Free Full Text]
  17. Pai R, Soreghan B, Szabo IL, Pavelka M, Baatar D, Tarnawski AS. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nat Med 2002; 8: 289–93.[CrossRef][Medline]
  18. Harizi H, Juzan M, Pitard V, Moreau JF, Gualde N. Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions. J Immunol 2002; 168: 2255–63.[Abstract/Free Full Text]
  19. Sharma S, Stolina M, Lin Y, et al. T cell-derived IL-10 promotes lung cancer growth by suppressing both T cell and APC function. J Immunol 1999; 163: 5020–8.[Abstract/Free Full Text]
  20. Stolina M, Sharma S, Lin Y, et al. Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immunol 2000; 164: 361–70.[Abstract/Free Full Text]
  21. Lanzavecchia A, Sallusto F. Dynamics of T lymphocyte responses: intermediates, effectors, and memory cells. Science 2000; 290: 92–7.[Abstract/Free Full Text]
  22. Pradelles P, Grassi J, Maclouf J. Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay. Anal Chem 1985; 57: 1170–3.[Medline]
  23. Maxey KM, Maddipati KR, Birkmeier J. Interference in enzyme immunoassays. J Clin Immunoassay 1992; 15: 116–20.
  24. Goodwin JS, Bankhurst AD, Messner RP. Suppression of human T-cell mitogenesis by prostaglandin. Existence of a prostaglandin-producing suppressor cell. J Exp Med 1977; 146: 1719–34.[Abstract/Free Full Text]
  25. Kabashima K, Sakata D, Nagamachi M, Miyachi Y, Inaba K, Narumiya S. Prostaglandin E2-EP4 signaling initiates skin immune responses by promoting migration and maturation of Langerhans cells. Nat Med 2003; 9: 744–9.[CrossRef][Medline]
  26. Finke JH, Zea AH, Stanley J, et al. Loss of T-cell receptor zeta chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res 1993; 53: 5613–6.[Abstract/Free Full Text]
  27. Ghosh P, Sica A, Young HA, et al. Alterations in NF kappa B/Rel family proteins in splenic T-cells from tumor-bearing mice and reversal following therapy. Cancer Res 1994; 54: 2969–72.[Abstract/Free Full Text]
  28. Appleman LJ, Tzachanis D, Grader-Beck T, van Puijenbroek AA, Boussiotis VA. Helper T cell anergy: from biochemistry to cancer pathophysiology and therapeutics. J Mol Med 2001; 78: 673–83.[CrossRef][Medline]
  29. Schmielau J, Finn OJ. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res 2001; 61: 4756–60.[Abstract/Free Full Text]
  30. Schmielau J, Nalesnik MA, Finn OJ. Suppressed T-cell receptor zeta chain expression and cytokine production in pancreatic cancer patients. Clin Cancer Res 2001; 7 (3 Suppl): 933s–939s.[Medline]
  31. Nieland JD, Loviscek K, Kono K, et al. PBLs of early breast carcinoma patients with a high nuclear grade tumor unlike PBLs of cervical carcinoma patients do not show a decreased TCR zeta expression but are functionally impaired. J Immunother 1998; 21: 317–22.[Medline]
  32. Lucey DR, Clerici M, Shearer GM. Type 1 and type 2 cytokine dysregulation in human infectious, neoplastic, and inflammatory diseases. Clin Microbiol Rev 1996; 9: 532–62.[Abstract]
  33. Onishi T, Ohishi Y, Goto H, Tomita M, Abe K. An assessment of the immunological status of patients with renal cell carcinoma based on the relative abundance of T-helper 1- and -2 cytokine-producing CD4+ cells in peripheral blood. BJU Int 2001; 87: 755–9.[CrossRef][Medline]
  34. Duan Z, Feller AJ, Penson RT, Chabner BA, Seiden MV. Discovery of differentially expressed genes associated with paclitaxel resistance using cDNA array technology: analysis of interleukin (IL) 6, IL-8, and monocyte chemotactic protein 1 in the paclitaxel-resistant phenotype. Clin Cancer Res 1999; 5: 3445–53.[Abstract/Free Full Text]
  35. Kunz M, Toksoy A, Goebeler M, Engelhardt E, Brocker E, Gillitzer R. Strong expression of the lymphoattractant C-X-C chemokine Mig is associated with heavy infiltration of T cells in human malignant melanoma. J Pathol 1999; 189: 552–8.[CrossRef][Medline]
  36. Baggiolini M, Dewald B, Moser B. Human chemokines: an update. Annu Rev Immunol 1997; 15: 675–705.[CrossRef][Medline]
  37. Groux H, Bigler M, de Vries JE, Roncarolo MG. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J Exp Med 1996; 184: 19–29.[Abstract/Free Full Text]
  38. Letterio JJ, Roberts AB. Regulation of immune responses by TGF-beta. Annu Rev Immunol 1998; 16: 137–61.[CrossRef][Medline]
  39. McCoy JM, Wicks JR, Audoly LP. The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis. J Clin Invest 2002; 110: 651–8.[CrossRef][Medline]
  40. Menetrier-Caux C, Montmain G, Dieu MC, et al. Inhibition of the differentiation of dendritic cells from CD34(+) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor. Blood 1998; 92: 4778–91.[Abstract/Free Full Text]
  41. Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 2000; 192: 1213–22.[Abstract/Free Full Text]
  42. Bell D, Chomarat P, Broyles D, et al. In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J Exp Med 1999; 190: 1417–26.[Abstract/Free Full Text]
  43. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Ann Rev Immunol 2003; 21: 685–711.[CrossRef][Medline]
  44. Roper RL, Phipps RP. Prostaglandin E2 regulation of the immune response. Adv Prostaglandin Thromboxane Leukot Res 1994; 22: 101–11.[Medline]
  45. Snijdewint FG, Kalinski P, Wierenga EA, Bos JD, Kapsenberg ML. Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes. J Immunol 1993; 150: 5321–9.[Abstract]
  46. Misra N, Selvakumar M, Singh S, et al. Monocyte derived IL 10 and PGE2 are associated with the absence of Th 1 cells and in vitro T cell suppression in lepromatous leprosy. Immunol Lett 1995; 48: 123–8.[CrossRef][Medline]
  47. Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavecchia A, Alber G. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med 1996; 184: 747–52.[Abstract/Free Full Text]
  48. Kalinski P, Vieira PL, Schuitemaker JH, de Jong EC, Kapsenberg ML. Prostaglandin E(2) is a selective inducer of interleukin-12 p40 (IL-12p40) production and an inhibitor of bioactive IL-12p70 heterodimer. Blood 2001; 97: 3466–9.[Abstract/Free Full Text]
  49. Morecki S, Yacovlev E, Gelfand Y, Trembovler V, Shohami E, Slavin S. Induction of antitumor immunity by indomethacin. Cancer Immunol Immunother 2000; 48: 613–20.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
J. E. Talmadge
Pathways Mediating the Expansion and Immunosuppressive Activity of Myeloid-Derived Suppressor Cells and Their Relevance to Cancer Therapy
Clin. Cancer Res., September 15, 2007; 13(18): 5243 - 5248.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. D. Basu, T. L. Tinder, J. M. Bradley, T. Tu, C. L. Hattrup, B. A. Pockaj, and P. Mukherjee
Cyclooxygenase-2 Inhibitor Enhances the Efficacy of a Breast Cancer Vaccine: Role of IDO
J. Immunol., August 15, 2006; 177(4): 2391 - 2402.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. M. Chemnitz, J. Driesen, S. Classen, J. L. Riley, S. Debey, M. Beyer, A. Popov, T. Zander, and J. L. Schultze
Prostaglandin E2 Impairs CD4+ T Cell Activation by Inhibition of lck: Implications in Hodgkin's Lymphoma
Cancer Res., January 15, 2006; 66(2): 1114 - 1122.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. J. McKallip, M. Nagarkatti, and P. S. Nagarkatti
{Delta}-9-Tetrahydrocannabinol Enhances Breast Cancer Growth and Metastasis by Suppression of the Antitumor Immune Response
J. Immunol., March 15, 2005; 174(6): 3281 - 3289.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
L A Emens, R T Reilly, and E M Jaffee
Breast cancer vaccines: maximizing cancer treatment by tapping into host immunity
Endocr. Relat. Cancer, March 1, 2005; 12(1): 1 - 17.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pockaj, B. A.
Right arrow Articles by Mukherjee, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pockaj, B. A.
Right arrow Articles by Mukherjee, P.
Related Collections
Right arrow Other Breast

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH