Department of Surgery, Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri
Correspondence: T. Mohanakumar, Ph.D., Washington University School of Medicine, Department of Surgery and Pathology, Box-8109, 3328 CSRB, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail: kumart{at}msnotes.wustl.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) produced after DC activation was responsible for the antitumor activity of DC. Interferon-
, interleukin-15, or LPS treatment of DC markedly augmented the effector function of DC against most of the breast cells, indicating heterogeneity of the tumor and its susceptibility to cytokine-mediated damage. Treatment of LPS-activated DC or cell-free supernatant with anti-human TNF- significantly reduces the antitumor effect against the tumor cells tested. These results suggest that in addition to their predominant role as immune regulatory cells, DC could serve as innate effector cells in tumor immunity.
Key Words: apoptosis • TNF- • IFN- • LPS
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The immune surveillance role of DC has been documented in several systems, which involves the secretion of important immunoregulators such as tumor necrosis factor (TNF-) [14
]. DC are found to infiltrate to the areas surrounding human solid tumors, and the density of their infiltration has been correlated with the condition of the disease [15
16
17
]. Besides the classical antigen-presenting role of DC to T cells, the direct role of DC against tumor has been reported in recent times [18
19
20
21
].
In this communication, we studied the direct interaction of human DC to a variety of breast cancer cell lines. DC showed variable levels of cytotoxicity as well as growth inhibition of breast cancer cell lines. Pretreatment of DC with interferon- (IFN-) and interleukin-15 (IL-15) or lipopolysaccharide (LPS) augmented DC-mediated growth inhibition and cytotoxicity. This antitumor effect of DC is associated with apoptosis of tumor cells and provides a new mechanism by which DC mediate antitumor function in breast cancer.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-15, and IFN- as well as all antibodies used in this study were purchased from Pharmingen BD Biosciences (San Diego, CA). LPS was purchased from Sigma Chemical Co. (St. Louis, MO).
Cells
The MCF-7 (breast adenocarcinoma), BT-20 (breast carcinoma), HBL-100 (breast and lung carcinoma), MDA-MB-231 (breast adenocarcinoma), MDA-MB-415 (breast adenocarcinoma), BT-474 (breast ductal carcinoma), MDA-MB-175 (breast ductal carcinoma), and MRC-5 (fibroblast) cell lines were purchased from American Type Culture Collection (Manassas, VA). B-lymphoblastoid cell lines (B-LCL) immortalized by Epstein-Barr virus were established in the laboratory. The cell culture medium used was RPMI 1640 (Life Technologies, Grand Island, NY), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), and 10 µg/ml ciprofloxacin.
Isolation of DC
Peripheral blood DC were purified from peripheral blood mononuclear cells (PBMC) by incubating the adherent cell population overnight at 37°C, 5% CO2. The peripheral blood DC generally adhere along with the monocytes but become nonadherent after overnight incubation. The DC population was isolated from this heterogeneous, nonadherent population of cells by treatment with monoclonal antibodies (mAb) to CD3, CD14, CD19, and CD56 plus goat anti-mouse immunoglobulin (Ig)-conjugated magnetic beads (Dynal, Oslo, Norway). After four rounds of treatment, DC were purified from a harvested, nonadherent population. The purity of DC was determined by surface expression of DC-specific markers such as CD1a, CD11c, CD83, and human leukocyte antigen (HLA)-DR and was analyzed in a FACScan (Becton Dickinson, San Jose, CA). The purity of DC generated by this method was >98% and was free from monocytes and other contaminating leukocytes. Cell viability was >98% as determined by trypan blue dye exclusion.
The enriched DC show surface expression of CD11c, CD1a, CD83, and HLA-DR and do not express CD3, CD14, CD19, or CD56, indicating effector cells used for the functional analysis are blood DC. Figure 1 represents the surface phenotype of one DC preparation used in the study. Up-regulation of costimulatory molecules (CD40, CD80, CD86), CD83, and HLA-DR was observed in DC after incubation with indicated cytokines or LPS, and the expression of CD11c and CD1a remains unchanged (data not shown). The expression of CD83 in our DC preparation is similar to Fanger et al. [19 ], who also observed that fresh human peripheral blood-derived DC start expressing CD83 after overnight incubation with media.
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Figure 1. The phenotype of human peripheral blood-derived DC. PBMC were adhered for 2–4 h at 37°C, 5% CO2. The nonadherent cells were removed, and adherent cells were washed and incubated at 37°C, 5% CO2, for another 18–24 h in complete medium. After overnight incubation, the DC were isolated from the loosely adherent cell population by depleting CD3+, CD14+, CD19+, and CD56+ cells using specific mAb and goat anti-mouse, polyvalent Ig-coated immunomagnetic beads. Expression of the indicated antigens was analyzed by single color flow cytometry using PE- or FITC-conjugated mAb. Data represent one individual donor. Similar results were obtained from other unrelated donors used in the study.
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(500 pg/ml), IFN- (1000 U/ml), IL-15 (200 pg/ml), GM-CSF (1000 U/ml), or LPS (10 µg/ml). After 24 h, the cells were washed (x3), and tumor cells (5x103) were added to the wells. Plates were incubated at 37°C, 5% CO2, for 24 h and then pulsed with 1 µCi/well of [3H]thymidine (ICN, Irvine, CA). The plates were harvested, and thymidine incorporation was assessed by means of liquid scintillation counter (Wallac, Gaithersburg, MD). The data are presented as the percentage of inhibition calculated from the following formula: % Inhibition = (1-test cpm/control cpmx100), where test cpm is thymidine incorporation by tumor cells cultured with DC after various stimulations, and control cpm is the corresponding value of tumor cell only cultured in the absence of DC. DC with or without various stimuli did not incorporate a significant amount of radioactivity (less than 700), and the tumor cells usually incorporated 7500–85,000 cpm, depending on the type of tumor line.
In some experiments, DC were plated in 96-well plates and incubated with or without LPS (10 µg/ml), IFN- (1000 U/ml), or IL-15 (200 pg/ml) for 24 h. After 24 h, the supernatant was collected and cells were washed with complete medium (x3). The cells were treated with a saturating concentration of antibodies-to-human TNF-, human IL-12 (p40/p70), or human IFN- for 1 h before addition of tumor cells at an effector:target (E:T) ratio of 10:1. The supernatant generated from DC after LPS activation was added to separate 96-well plates and treated with antibodies to human TNF- or human IL-12 (p40/p70) for 1 h before addition of tumor cells. The incubation of tumor cells with DC alone or DC-derived supernatant was the same as before. The cells were pulsed with [3H]thymidine for the last 24 h of incubation before being harvested.
Intracellular staining of TNF- in DC
Peripheral blood-derived DC were cultured for 24 h with media alone or media supplemented with TNF-, GM-CSF, IFN-, IL-15, or LPS. The cells were washed and fixed with 1% paraformaldehyde for 5 min at room temperature followed by treatment with -gluco pyranoside (7 mg/ml) for 5 min at room temperature. The cells were washed and stained with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human TNF- for 45 min and analyzed in a FACScan.
DC cytotoxic activity assay
The lytic activity of peripheral blood DC was measured by 18-h 51Cr-release assay. DC were cultured for 24 h in media alone or in the presence of GM-CSF (1000 U/ml), TNF- (500 pg/ml), IFN- (1000 U/ml), IL-15 (200 pg/ml), or LPS (10 µg/ml) in 96-well plates at 37°C, 5% CO2. The cells were washed (x3) and resuspended in complete media before addition of radiolabeled tumor cells. Tumor cells, MRC-5, or B-LCL were labeled with 100 µCi 51Cr for 1 h at 37°C, washed three times, and resuspended in complete media. Target cells (5x103) were added to the unstimulated and cytokine-stimulated DC at a fixed E:T ratio of 10:1 and incubated for 18 h at 37°C, 5% CO2. After incubation, the supernatant was collected, and 51Cr release was measured in a counter. Percent-specific lysis was determined using the following formula: (experimental cpm-spontaneous cpm/maximum cpm-spontaneous cpm) x 100.
Detection of apoptosis
We evaluated the ability of unactivated or activated DC to induce apoptotic cell death in breast cancer cell lines by binding FITC-conjugated Annexin V. Light-scatter characteristics were used to distinguish the tumor cells from the DC, such that only the tumor cells were counted in the analysis. After 8 h of incubation, the percentage of FITC-conjugated Annexin V-positive cells was analyzed by flow cytometry (Becton Dickinson). In some experiment, the incubation of tumor cells and various DC populations was extended for 24 h followed by staining with Annexin V and propidium iodide (PI) and was analyzed by flow cytometry.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-15, or LPS significantly inhibited the growth of MCF-7 cells by 25%, 22%, and 36%, respectively. On the other hand, unstimulated DC could inhibit breast cancer cell growth by only 7%, which was not enhanced when tumor cells were treated with DC stimulated with TNF- or GM-CSF. Pretreatment of DC with recombinant human TNF- or GM-CSF had no significant, stimulatory capacity on DC as compared with the effect of other cytokines and stimulatory agents (Fig. 2)
.
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Figure 2. DC-mediated growth inhibition of human breast tumor lines in vitro. DC (5x104)/well were cultured in 96-well plates with or without indicated cytokines or LPS at 37°C, 5% CO2. At 24 h, the cells were washed (x3) with complete medium and resuspended in the same medium. Indicated tumor lines MCF-7 (A), HBL-100 (B), MDA-MB-231 (C), MDA-MB-415 (D), LCL, or MRC-5 at 5 x 103 cells/well were added to the wells containing naïve or activated DC and cocultured for 24 h at 37°C, 5% CO2, and for an additional 24 h, with 1H3 thymidine before being harvested. The results are presented as the mean percentage of inhibition of tumor cell proliferation ± SD of triplicate wells. Thymidine incorporation into DC alone was less than 700 cpm. Representative of five experiments is shown here.
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, IL-15, or LPS for 24 h. The coculture of activated DC and tumor cells was treated with a saturating concentration of anti-human TNF-, anti-human IL-12, or anti-human IFN- antibody and was incubated for another 24 h prior to the addition of thymidine. Treatment of activated DC with anti-human TNF-, but not with anti-human IL-12 or anti-human IFN-, abolishes the antitumor potential of cytokine- or LPS-activated DC on MCF-7, HBL-100, MDA-MB-231, or MDA-MB-415 breast cancer cell lines. Figure 3
represents the growth inhibition of tumor cells by LPS-activated DC. Similar results were obtained with DC preparation after activation with IFN- and IL-15 (data not shown). These results demonstrate that TNF- produced by activated DC may contribute to growth inhibition of breast cancer cell lines.
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Figure 3. Enhancement effect of antitumor potential of LPS-activated DC is mediated by soluble factor. DC (5x104/well) were incubated with or without LPS (10 µg/ml). At 24 h, the supernatants were collected, and DC were washed (x3) with complete medium and treated with anti-human TNF-, anti-human IL-12, or anti-human IFN- at a neutralizing concentration for 1 h before the addition of respective tumor targets. The DC and tumor cells MCF-7 (A), HBL-100 (B), MDA-MB-231 (C), or MDA-MB-415 (D) were incubated for 24 h at 37°C, 5% CO2, and for an additional 24 h, with 1H3 thymidine before being harvested. The results are presented as the mean percentage of inhibition of tumor cell proliferation ± SD of triplicate determination. Representative of four experiments is shown here.
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-mediated growth inhibition by activated DC was further supported by increased growth inhibition of breast cancer cells by cell-free culture supernatant derived from DC after LPS treatment. Treatment of this supernatant (pretreated with polymyxin B to inactivate the LPS) with anti-human TNF- and not by anti-human IL-12 significantly inhibits the tumor cell growth. The breast cancer cell and the DC-derived supernatant were incubated in the same way before being pulsed for determination of proliferation of individual tumor cell lines (Fig. 4
). These data suggest that TNF- derived from activated DC could acts as a potential antitumor molecule, which significantly inhibits the growth of breast cancer cells. Supernatants generated from DC after treatment with IFN- or IL-15 were also studied after neutralizing the endogenous IFN- or IL-15 with mAb specific to indicated cytokine before used for tumor growth inhibition study (data not shown).
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Figure 4. Contact independent inhibition of tumor targets by soluble factors derived from LPS-activated DC. The cell-free culture supernatants generated from DC cultured with media alone (DC1Sup) or with LPS (DC2Sup) were filtered by a membrane with 0.45 µm pore size. The undiluted, LPS-activated, DC-derived supernatants were further treated with anti-human TNF- (DC3Sup) or anti-human IL-12 (DC4Sup) antibody for 1 h in 96-well plates before the addition of respective tumor targets MCF-7 (A), HBL-100 (B), MDA-MB-231 (C), or MDA-MB-415 (D). The tumor cells were incubated with the supernatant at 37°C, 5% CO2, for 24 h and for an additional 24 h, with 1H3 thymidine before being harvested. The results are presented as the mean percentage inhibition ± SD of triplicate determination. Representative of four experiments is shown here.
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has been documented in DC after stimulation with IFN-, IL-15, or LPS. TNF- also induces a low level of TNF- in DC. Figure 5
represents intracellular TNF- production in DC stimulated with media alone or indicated cytokines or LPS. The light-colored histogram represents the isotype control, and the overlayed, dark-colored histogram represents the staining with TNF-.
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Figure 5. Intracellular staining of TNF- in DC after treatment with cytokines or LPS. Blood DC were cultured with media alone or media supplemented with GM-CSF, TNF-, IFN-, IL-15, or LPS for 24 h. The DC were fixed with 1% p-HCHO (para-formaldehyde) for 5 min at room temperature followed by washing, and fixed with -gluco pyranoside for 5 min at room temperature. The fixed, permeabilized cells were stained with mouse anti-human TNF- for 45 min on ice and analyzed by fluorescein-activated cell sorter (FACS) analysis. The light-colored histogram represents the isotype control, and the overlayed, dark histogram represents the TNF- staining in indicated treatment. Representative of four experiments is shown here.
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, IL-15, or LPS show enhanced lysis compared with DC alone or DC treated with GM-CSF. Lysis of MCF-7 cell lines was augmented from <5% by DC alone to as much as 30% in the presence of DC pretreated with LPS (Fig. 6A
). Similar results were observed with three other cell lines tested (HBL-100, MDA-MB-231, and MDA-MB-415). No significant enhancement of lysis was observed on tumor cells after coculture with TNF-- or GM-CSF-activated DC (Fig. 6A
6B
6C
6D)
. DC-mediated cytotoxicity of breast cancer cell lines appears to be specific, as the LCL and MRC-5 as well as three other breast cancer cell lines showed no significant lysis by unstimulated as well as by activated DC (Table 1
). The spontaneous release varies from 10–18% among the tumor targets tested.
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Figure 6. Cytolytic activity of human DC against breast cancer cell lines. DC were cultured alone or in the presence of recombinant human TNF-, recombinant human GM-CSF, recombinant human IFN-, recombinant human IL-15, or LPS for 24 h at 37°C, 5% CO2, in 96-well plates. After extensive washing, 5 x 103 51Cr-labeled MCF-7 (A), HBL-100 (B), MDA-MB-231 (C), or MDA-MB-415 (D) was added into wells at an E:T ratio of 10:1 and incubated for 18 h at 37°C, 5% CO2. The supernatants were collected and released; 51-labeled Cr was measured in a radioactive counter. The results are presented as the mean ± SD of triplicate wells. The experiment was repeated five times with unrelated donors and produced similar results.
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Table 1. Tumoricidal Activity of Cytokine-Stimulated, Peripheral Blood-derived DC
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- or IL-15-treated DC also shows higher Annexin V-positive cells compared with the treatment with unactivated DC (Fig. 7A
). Similar results were observed in other cell lines tested. The differences in Annexin positivity among the tumor cell lines could be attributed to the wide biological differences in the tumor cell line and unknown DC-tumor interaction. HBL-100 was particularly susceptible to DC-mediated apoptosis. The percent apoptosis was found to be higher in this cell line compared with others tested (Fig. 7B)
. This result was also tallied with the increased susceptibility of HBL-100 to DC-mediated growth inhibition and cytotoxicity. Two other cell lines also showed enhanced apoptosis in the presence of activated DC (Fig. 7C and 7D)
.
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Figure 7. Breast tumor targets undergo enhanced apoptotic cell death by activated DC. Tumor cell targets undergo significant apoptotic cell death when cultured with IFN--, IL-15-, or LPS-stimulated DC as determined by phosphatidyl serine externalization. MCF-7 (A), HBL-100 (B), MDA-MB-231 (C), or MDA-MB-415 (D) tumor cells were cultured for 8 h in complete medium alone, in the presence of unstimulated DC, or with GM-CSF (DC1)-, IFN- (DC2)-, IL-15 (DC3)-, or LPS-stimulated DC (DC4) at an E:T ratio 10:1. Cells were then stained with FITC-Annexin V and analyzed by flow cytometry. The percentage of Annexin V-positive tumor cells is indicated for each condition. Histograms represent 104-gated tumor cells. The experiment was repeated two times using two different donors with identical results.
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Figure 8. Enhanced apoptotic cell death in tumor cells by DC upon longer incubation. Similar to Figure 7
, the tumor cell HBL-100 was cultured with unactivated or activated DC for 24 h at an E:T ratio of 10:1. (A) The histogram presentation of Annexin V-positive cells upon treatment with unactivated or activated DC. (B) The double staining of the tumor cells with Annexin V and PI for demonstration of apoptotic as well as dead cells. Light scatter characteristics were used to distinguish the tumor cells from the DC, such that only the tumor cells were counted in the analysis. The experiment was repeated two times with similar results.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, DC derived from the peripheral blood were used. DC demonstrated typical phenotypes including CD1a, CD11c, and CD83. There was no contamination (<2%) with T cells (CD3), B cells (CD19), and natural killer cells (CD56) in the DC population used, indicating that the effector function presented in this study is mediated by DC. The expression of mature DC marker CD83 in our DC preparation was also supported by the similar observation made by Fanger et al. [19
]. Our results demonstrate that activated human peripheral blood DC have enhanced cytostatic as well as a cytotoxic role against the breast tumor cell lines, which are initiated by the apoptosis of tumor cells. The tumor growth inhibition by DC was significantly enhanced by pretreatment of DC with IFN-, IL-15, or LPS. Conversely, pretreatment with TNF- or GM-CSF had no significant effect (Fig. 2)
. We have observed that DC from all the donors have clear cytotoxic and cytostatic potential against the tumor cells after activation, although the degree of effector function varies among the donors and the tumor lines tested. The differential sensitivity of tumor cells to DC indicates the complex nature of DC-tumor interactions as well as heterogeneity of the individual tumor line. LPS-induced DC activation and its antitumor function are partially mediated through the participation of TNF- and not by IL-12 or IFN- (Fig. 3) . Similar observation was made with IFN-- or IL-15-activated DC (data not shown). Cell-free culture supernatant of LPS-activated DC also showed antitumor property, which is found to be inhibited by the pretreatment of supernatant with anti-TNF- and not by anti-IL-12 (Fig. 4)
. As DC preparation from peripheral blood was absolutely free from monocytes, induction of TNF- in DC is possibly mediated through the participation of Toll-like receptor by LPS treatment [26
]. This observation is in agreement with the results by Chapoval et al. [21
], where they induced TNF- production in monocyte-derived DC after LPS treatment for the antitumor immunity. Activated DC from several donors is also cytotoxic to a number of breast cancer cell lines (Fig. 6 , Table 1
). There was no significant cytotoxicity against the B-LCL or the MRC-5 fibroblast line (Table 1)
.
Another important finding is that breast cancer cell lines cultured with activated DC underwent apoptosis. Apoptosis was noted as early as 8 h, indicating its role in breast cancer cell growth inhibition as well as lysis noted in our study. The degree of apoptosis was further enhanced upon longer incubation of DC and tumor cells (Fig. 8) . It has been shown that tumor-derived apoptotic bodies can be taken up, processed, and presented to CD8+ T cells [4 , 5 ]. The result presented in our study strongly suggests that DC may play an important role in this process.
In summary, the results presented herein indicate an important role of DC in host resistance against breast cancer. Peripheral blood DC induced apoptosis of several breast cancer cell lines and resulted in growth inhibition as well as lysis of breast cancer cells. These proactive roles of DC strongly suggest an important regulatory effector function for DC in the tumor microenvironment, which could prevent growth and metastasis of breast cancer. Taken together, we propose that drug-induced mobilization of DC in and around tumor sites may have a beneficial effect against the progression of the disease by the direct effector functions reported in this communication as well as the well-characterized tumor antigen presentation capability of DC.
Received December 17, 2001; revised February 5, 2002; accepted February 15, 2002.
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