Dysfunction of Murine Dendritic Cells Induced by Incubation with ...

Cellular & Molecular Immunology

133

Article

Dysfunction of Murine Dendritic Cells Induced by Incubation with Tumor Cells Fengguang Gao1, 2, Xin Hui1, Xianghuo He1, Dafang Wan1 and Jianren Gu1, 3 In vivo studies showed that dendritic cell (DC) dysfunction occurred in tumor microenvironment. As tumors were composed of many kinds of cells, the direct effects of tumor cells on immature DCs (imDCs) are needed for further studies in vitro. In the present study, bone marrow-derived imDCs were incubated with lymphoma, hepatoma and menaloma cells in vitro and surface molecules in imDCs were determined by flow cytometry. Then, imDCs incubated with tumor cells or control imDCs were further pulsed with tumor lysates and then incubated with splenocytes to perform mixed lymphocyte reaction. The DC-dependent tumor antigen-specific T cell proliferation, and IL-12 secretion were determined by flow cytometry, and enzyme-linked immunosorbent assay respectively. Finally, the DC-dependent tumor-associated antigen-specific CTL was determined by enzyme-linked immunospot assay. The results showed that tumor cell-DC incubation down-regulated the surface molecules in imDCs, such as CD80, CD54, CD11b, CD11a and MHC class II molecules. The abilities of DC-dependent antigen-specific T cell proliferation and IL-12 secretion were also decreased by tumor cell incubation in vitro. Most importantly, the ability for antigenic-specific CTL priming of DCs was also decreased by incubation with tumor cells. In the present in vitro study demonstrated that the defective abilities of DCs induced by tumor cell co-incubation and the co-incubation system might be useful for future study of tumor-immune cells direct interaction and for drug screen of immune-modulation. Cellular & Molecular Immunology. 2008;5(2):133-140.

Key Words: immunity, dendritic cell, dysfunction, co-culture, tumor

Introduction It is well established that tumor cells express many kinds of antigens which could be recognized by dendritic cells (DCs), subsequently activating the tumor-specific T-cell response (1). However, this response in fact does not usually occur in most types of human cancer or in animal models, indicating that the dysfunction of host immune system including DCs might be one of the main mechanisms by which tumors escape from host immune control (2-6), as well as an important issue to limit the success of cancer immunotherapy (7). In fact, suppressed DC functions have been reported in various models simulating the tumor microenvironment, and in

1 The National Laboratory for Oncogenes and Related Genes, Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China; 2

School of Medicine, Xiamen University, Xiamen 361005, China;

3

Corresponding to: Dr. Jianren Gu, The National Laboratory for Oncogenes and Related Genes, Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China. E-mail: [email protected] Received, Mar 15 2008. Accepted Apr 7, 2008. Copyright © 2008 by The Chinese Society of Immunology Volume 5

tumor-bearing animals, as well as in cancer patients (8-10). Recently, an in vitro incubation system including irradiated tumor cells, DCs, CD4+CD25- T cells was also reported to induce the expansion of human T regulatory type 1 cells (11). All these co-incubation systems include relative tumor supernatants, soluble tumor-derived factors, irradiated tumor cells, using 0.4 μm diameter pore membrane to observe the soluble factor effect on DC function. But, until now, little is known about the effects of tumor cells on immature DCs (imDCs) by direct tumor cell-imDC contact. Therefore, in vitro studies are urgently needed to explore the effects of live tumor cells on imDCs based on direct tumor cell-DC co-incubation. In the present study, using 8.0-12 μm diameter pore co-incubation system, which permits tumor cells to directly contact on DCs, we co-incubated imDCs with different original tumor cells to study the effect of live tumor cells contact on imDCs. We firstly demonstrated that tumor cell-imDC co-incubation down-regulated surface molecules on imDCs. Secondly, we illustrated that the abilities of DCs in T cell priming and IL-12 secretion were also decreased by such tumor cell-imDC co-incubation. Finally and most importantly, we revealed that the ability of DCs to prime tumor antigenic-specific CTL was obviously decreased by such in vitro co-incubation. Our study indicated that myeloid DC function defects do occur in tumor cell-DC co-incubation system, which might be valuable for studying the tumor-

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Dysfunction of Murine Dendritic Cells Induced by Incubation with Tumor Cells

immune cells direct interaction and for future immunemodulation drug screening.

Materials and Methods Reagents GM-CSF and IL-4 were obtained from R&D (Minneapolis, USA). BD TrucountTM Tubes were obtained from BD Biosciences (San Jose, USA). Transwell™ 8.0 and 12 μm diameter polycarbonate membrane was obtained from Corning Headquarters (New York, USA). Fluorescene conjugated antibodies were obtained from eBioscience (San Diego, USA). Mouse IL-12 ELISA Kit was obtained from Bender MedSystems (Vienna, Austria), and IFN-γ ELISPOT Kit was from U-CyTech Biosciences (Utecht, Netherlands). The H-2Kb CTL peptide of ovalbumin (SIINFEKL) was synthesized by Sangong (Shanghai, China). Animals Pathogen-free C57BL/6 mice (female, 6-8 weeks old) were bought from Shanghai Laboratory Animal Center of Chinese Academy of Sciences (China) and kept at the Animal Center of Cancer Institute, Shanghai Jiao Tong University. All animal studies were approved by the Review Board, Cancer Institute, Shanghai Jiao Tong University. Cell lines EG7 cell line which carries a complete copy of chicken ovalbumin (OVA) mRNA and the neomycin (G418) resistance gene, was derived from the C57BL/6 (H-2b) mouse lymphoma cell line EL4 (ATCC TIB-39) (12). EG7 cells were cultured in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1.0 mM sodium pyruvate and supplemented with 0.05 mM 2-mercaptoethanol and 0.4 mg/ml G418, 10% FBS (Invitrogen Life Technologies). Hepa 1-6 cell line was a derivative of the BW7756 mouse hepatoma that arose in a C57BL/6 mouse (ATCC CRL-1830) (13). B16 cell line was derived from C57BL/6 mouse skin melanoma (ATCC CRL-6475) (14). Hepa 1-6 and B16 cells were cultured in Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 10% FBS. EG7 and Hepa 1-6 cell lines were obtained from Dr. Cao XT (Second Military Medical University, China). B16 cell line was a kindly gift of Dr. Yao M (Shanghai Jiao Tong University, China). Preparation of tumor cell lysate Tumor cell lysate was prepared as previously described (15). Briefly, Hepa 1-6 and B16 cells cultured in Dulbecco’s modified Eagle’s medium were harvested using trypsin buffer. EG7 cells, which were cultured in CM, were harvested directly. After two washes with cold PBS, the cell pellet was then resuspended in lysis buffer (20 mM HEPES buffer, 50 mM NaCl, 10 mM KCl, 1 mM EDTA, 200 mM sucrose, pH 7. 4) at a concentration of 2 × 106 cells/250 μl. Volume 5

Tumor cells were then subjected to repeat (6×) freezing at -80°C and thawing at 37°C. After the last thaw, cells were sonicated for 10 s/ml of lysate and centrifuged at 14,000 rpm for 20 min at 4°C. Supernatant was then collected into vials and stored at -80°C. Bone marrow-derived murine DCs Bone marrow-derived imDCs were prepared as previously described (16). Briefly, bone marrow mononuclear cells were prepared from bone marrow suspensions by depletion of red cells and then were cultured at a density of 1 × 106 cells/ml in RPMI 1640 complete medium with 10 ng/ml of GM-CSF and 1 ng/ml of IL-4. Non-adherent cells were gently washed out on day 4 of culture; the remaining loosely adherent clusters were used as imDCs which were further coincubated with tumor cells. DCs co-incubation with tumor cells To observe the effects of co-incubation with tumor cells on imDCs surface molecules expression and the abilities of DC-dependent antigenic-specific CTL priming, Transwell™ 8.0-12 μm diameter polycarbonate membranes were used to incubate cells according to the methods previously described (17). Briefly, Transwell™ polycarbonate membranes were wet with media in a 6-well plate, turned upside down, 1 × 105 EG7, Hepa 1-6 or B16 cells were added to the outside membrane of the Transwell™ in 400 μl volume respectively and incubated in 37°C CO2 incubator for 4 hours to allow the cell attachment. Then, Transwell™ was gently turned over and 1 × 106 imDC cells were seeded to the inside of the Transwell™. Media was put in the plastic well outside the Transwell™ and incubated for another 14 h. Control cultures containing 1 × 106 imDCs alone or pulsed with either 1 × 105 tumor cell lysate or ovalbumin 1 μg/ml were used as negative and positive controls. At the end of incubation, DCs were harvested and stained for flow cytometric measurement or pulsed with same amount tumor lysate and performed mixed lymphocyte reactions (MLRs). Flow cytometric measurement Expression of cell surface molecules was determined by flow cytometry according to the methods described previously (16). Before staining with relevant Abs, DCs were incubated for 15 min at 4°C with antibody to CD16/CD32 at a concentration of 1 μg per 1 × 106 cells for blockade of Fc receptors. Staining was performed on ice for 30 min and then cells were washed with ice-cold PBS, containing 0.1% NaN3 and 0.5% BSA. Analysis was performed using FACSCalibur and the mean fluorescene intensity (MFI) of each DCs population was analyzed with CellQuest software. Antigen-specific T cell proliferation assays Antigen-specific T cell proliferation assay was performed as described previously (17) and determined by BD Trucount system (16). Briefly, 1 × 106 imDCs were co-incubated with 1 × 105 live EG7 or Hepa 1-6 cells for 14 hours in the Transwell™ system. DCs without any treatment or pulsed with either same amount of tumor lysate or OVA 1 μg/ml

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were used as negative and positive controls. At the end of incubation, imDCs were harvested using trypsin buffer and then pulsed with 1 × 105 tumor lysate for another 4 hours. Then, DCs were added to plates containing 1 × 105 syngeneic splenocytes at a ratio of 1:10 to perform the MLRs. After 5-7 days incubation, supernatants of MLRs were collected for IL-12 determination by enzyme-linked immunosorbent assays (ELISA) and cells in MLRs were stained with antibodies in exactly 100 μl PBS. Thirty μl stained cells and 470 μl PBS were added to BD TrucountTM tubes and cellular data were acquired for 60 s with a flow cytometry. The number of T cells and control bead events acquired were analyzed, and the number of T cells in each well was calculated according to the formula: Numberabsolute count = (Number of events in region containing cell / Number of events in absolute count bead region) × (Number of beads per test / test volume).

A

CD80

15.45

Co-incubation with TSA cells down-regulates surface molecules on imDCs Co-stimulatory molecules on DCs are important for immunologic synapse formation, T cell activation and proliferation (1). EG7 cell line, which carries OVA mRNA and the neomycin (G418) resistance gene, is considered as tumor specific antigen (TSA) cell model (12). To explore the effects of tumor cell co-incubation on imDCs, imDCs were co-incubated with lymphoma EG7 cells in Transwell™ system and surface molecules on imDCs were determined by flow cytometry. As shown in Figure 1, the expression of surface molecules CD80, and CD86 had less been affected by lymphoma cells co-incubation (Figures 1A and 1B). But EG7 cells-DCs co-incubation significantly down-regulated CD40, Volume 5

6.46

CD40

CD54

41.20

39.12

35.86

26.75

20.43

29.25

CD11a

CD11b

9.76 8.18

86.37 101.67

40.62

6.74

MHC II 12.51 14.99 11.93

Tumor antigen-specific ELISPOT assay Tumor antigen-specific enzyme-linked immunospot assay (ELISPOT) was performed using published method (19). Briefly, plate was coated with anti-IFNγ antibody and cells of MLRs were harvested and immediately plated in wells of ELISPOT plates with 1 × 105 cells/well. Cells were stimulated with either lymphoma EG7 specific SIINFELK peptide at a final concentration of 1 μg/ml or 1 × 105/ml Hepa 1-6 cells lysate and incubated at 37°C for 16-20 hours. Then, after detective antibody was added, the substrate was added and the spots were counted.

Results

7.58 6.92

25.60

IL-12 ELISA assay To determine IL-12 in the supernatants of MLRs, ELISA was performed according to the methods described previously (18).

Fluorescence

B

175

OVA

EG7

150

Change of expression (% of control)

Statistical analysis All data were expressed as mean and standard error means. Statistical significance was tested using Student’s t test. Statistical differences were considered to be significant if p < 0.05.

CD86

11.43

**

125

*

* *

*

CD54

MHC II CD11a

100 75 50 25 0 CD80

CD86

CD40

CD11b

Figure 1. Expression profiles of surface molecules on imDCs co-incubated with lymphoma cells. (A) After induced for 4 days in vitro, 1 × 106 imDCs were co-incubated with 1 × 105 EG7 cells in Transwell™ system. DCs cultured alone or pulsed with ovalbumin 1 μg/ml were used as negative and positive controls. The FACS data of surface molecules on imDCs. Open dotted gray profiles denote isotype controls; close light gray denote OVA pulsed imDCs; open dotted lines denote imDCs control and open black lines denote EG7 co-incubated imDCs. The numbers in histograms indicated the MFI of each DC population. (B) Histographic presentation of surface molecules on imDCs. Data were given as mean ± SEM, n = 6. *p < 0.05, **p < 0.01, indicated for EG7 versus OVA, analyzed by Student’s t test. OVA, ovalbumin; EG7, lymphoma cells expressing ovalbumin.

CD54, CD11a, CD11b and MHC class II molecules on imDCs. In contrast with untreated DCs, the MFI of CD86, CD40, CD54, CD11a, CD11b and MHC class II molecules were decreased by 85%, 78.5%, 89%, 72.6%, 80.7% and

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A

CD80

C

CD86

CD80

12.82

39.90 23.03

12.13

16.54

CD54

11.80

CD40

51.66 18.54

25.94 14.96

CD11a

CD11b 50.66

24.67

CD11b 58.00

12.87

58.00

20.30

CD54

11.80

19.44

13.71 12.13

10.83

44.40

19.07

CD11a

12.13

12.20

24.67

11.96

16.07

17.36

20.30

CD40

CD86 28.64

48.09

15.38

87.82

92.52

31.99

Fluorescence

Hepa 1-6

225 200 175 150

*

*

D

Hepa 1-6 lysate *

125 100

*

75 50 25 0

B16

300

Change in expression (% of control)

Change in expression (% of control)

B

Fluorescence

250 200 *

150

*

*

100 50 0

CD80 CD86 CD40 CD54 CD11a CD11b

B16 lysate

CD80 CD86 CD40 CD54 CD11a CD11b

Figure 2. Expression profiles of surface molecules on imDCs co-incubated with hepatoma or melanoma cells. After induced for 4 days, 1 × 106 imDCs were co-incubated with either 1 × 105 hepatoma or melanoma cells in Transwell™ system. DCs cultured alone or pulsed with 1 × 105 tumor cell lysate were used as negative and positive controls. The numbers in histograms indicated the MFI of each DC population. (A) The surface molecule expression on imDCs co-incubated with hepatoma cells. Open dotted gray profiles denote isotype controls, close light gray denote Hepa 1-6 lysate pulsed imDCs, open dotted lines denote imDCs control and open black lines denotes Hepa 1-6 co-incubated imDCs. (B) Histographic presentation of surface molecules expression on imDCs co-incubated with hepatoma cells. Data were given as mean ± SEM, n = 6. *p < 0.05, indicated for Hepa 1-6 versus Hepa 1-6 lysate, analyzed by Student’s t test. (C)The surface molecule expression in imDCs co-incubated with melanoma. Open dotted gray profiles denote isotype controls close light gray denote B16 lysate pulsed imDCs, open dotted lines denote imDCs control and open black lines denote B16 co-incubated imDCs. (D) Histographic presentation of surface molecule expression on DCs co-incubated with melanoma. Data were given as mean ± SEM, n = 4. *p < 0.05, indicated for B16 versus B16 lysate, analyzed by Student’s t test.

99.3%, respectively (Figure 1B). As compared with OVA pulsed DCs control, EG7 cells incubation decreased the MFI of CD40, CD54, CD11a, CD11b and MHC class II molecules to 64.2%, 83.2%, 73%, 70% and 82.8%, respectively (Figure 1B). Co-incubation with TAA cells down-regulates surface molecules on imDCs As hepatoma Hepa 1-6 and melanoma B16 cells do not express TSA, we further incubated imDCs with either hepatoma or melanoma cells to explore the effects of surface Volume 5

molecule down-regulation on imDCs by such co-incubation. As shown in Figure 2, co-incubation with either hepatoma or melanoma cells decreased a panel molecules expression except for CD86 and CD40. On the contrary with untreated DCs control, the MFIs of CD80, CD86, CD40, CD54, CD11a, and CD11b were changed to 73.6%, 135.1%, 174.7%, 81.2%, 67% and 89.5% respectively by hepatoma coincubation (Figure 2B). As compared with hepatoma lysatepulsed DCs, the MFIs of CD80, CD54 and CD11a were down-regulated to 59.1%, 60.1% and 49.1% respectively by such hepatoma co-incubation (Figure 2B). But, when

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A 2500 2000

IL-12 (pg/ml)

Proliferred cells number

A * **

1500 1000

Control DC/EG7 DC/EG7 lysate DC/OVA

3500 3000


2500 2000

*

1500 1000

500

500

0

0

Treatment

Treatment

B

B

1400 2000 1500 Control DC/Hepa DC/Hepa lysate

1000 500

**

1200

*

IL-12 (pg/ml)

Proliferred cells number

**

4000

1000 800

*

600 400

Control DC/Hepa DC/Hepa lysate

200 0

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Treatment

Treatment

Figure 3. The ability of DC to prime T cell proliferation was decreased by co-incubation with lymphoma or hepatoma cells. imDCs (1 × 106) cultured for 4 days were co-incubated with either 1 × 105 lymphoma or hepatoma cells in Transwell™ system. DCs cultured alone or pulsed with either 1 × 105 tumor cell lysate or OVA were used as negative and positive controls. After co-incubation, DCs were pulsed with 1 × 105 tumor lysate for 4 hours and performed MLRs with syngeneic splenocyte at a ratio of 1:10. At the end of 5-7 days incubation, proliferated lymphocytes number was determined by flow cytometry. (A) DCs-dependent T cell priming was decreased by co-incubation with lymphoma cells. Data were given as mean ± SEM, n = 6, data were representative of three independent tests. *p < 0.001 indicated for EG7 versus EG7 lysate; **p < 0.0001 for EG7 versus OVA, analyzed by Student’s t test. (B) DC-dependent T cell priming was decreased by incubation with hepatoma cells. Data were given as mean ± SEM, n = 6; data were representative of three independent tests. *p < 0.01 indicated for Hepa versus Hepaly, analyzed by Student’s t test. Hepa, Hepa 1-6.

Figure 4. The ability of DCs to produce IL-12 was decreased by co-incubation with lymphoma or hepatoma cells. DCs (1 × 106) cultured for 4 days were co-incubated with either 1 × 105 lymphoma or hepatoma cells. After co-incubation, DCs were pulsed with 1 × 105 tumor cell lysate for 4 hours and performed MLRs. IL-12 in the supernatants was determined by ELISA. Data were given as mean ± SEM, n = 6. These data were representative of three independent tests, analyzed by Student’s t test. (A) IL-12 secretion was decreased by co-incubation with lymphoma cells. IL-12 in control was below the limit of detection. *p < 0.001 indicated for EG7 versus EG7 lysate; **p < 0.0001 for EG7 versus OVA. (B) IL-12 secretion was decreased by co-incubation with hepatoma cells. *p < 0.05 indicated for Hepa versus control; **p < 0.001 for Hepa versus Hepa lysate. Hepa, Hepa 1-6.

considering the results derived from lymphoma EG7 co-incubation expriment, hepatoma Hepa1-6 treatment up-regulated CD86 with MFI increment to 151.5% (Figure 2B). Melanoma B16 co-incubation also gave the similar results. Compared to melanoma lysate-pulsed DCs, the MFI of CD11b in DCs was decreased to 66.5% while the MFIs of CD86 and CD40 were up-regulated to about 157.8% and 270.7%, respectively (Figures 2C and 2D).

further incubated with splenocytes for MLRs. As shown in Figure 3, tumor cell co-incubation decreased DC ability for T cell priming. For instance, DCs co-incubated with EG7 cells could only achieve 400 proliferated cells, while EG7 lysate or OVA pulsed DCs could have about 1807 and 1462 cells, respectively, indicating that tumor cells co-incubation decreased DCs ability for T cell priming (Figure 3A). Meanwhile, a remarkable decrement on DCs-dependent T cell priming ability was observed by hepatoma co-incubation, as hepatoma lysate-pulsed DCs could get 1365 proliferated cells, compared with 70 cells induced by Hepa 1-6 coincubated DCs.

Co-incubation with tumor cells decreased DC ability to stimulate T cell proliferation DCs are the most efficient antigen presenting cells (APCs), capable of priming naïve T lymphocytes (20). To further determine tumor cell co-incubation effect on DCs for T cell priming, imDCs co-incubated with either lymphoma or hepatoma cells were pulsed with relative tumor lysate and

Tumor cell co-incubation decreased DC ability to produce IL-12 DCs play a crucial role in generation of tumor antigenspecific immune response (21). As IL-12 is one of the key mediators to promote Th1 T cell priming (22), IL-12 in supernatants of MLRs was determined to indicate the ability of DCs-dependent Th1 priming. As shown in Figure 4, IL-12

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in the presence of either EG7 specific SIINFELK peptide or hepatoma lysate. As shown in Figure 5, tumor cell co-incubation could substantially reduce DC ability to prime antigen-specific CTL. EG7 incubated DCs could only develop 59 spots in contrast to 123 and 221 spots of EG7 lysate or OVA pulsed DCs respectively (Figure 5A). The dysfunction of lymphoma incubated DCs for CTL priming was also confirmed in hepatoma incubation DCs with 3 and 95 spots in hepatoma incubated or hepatoma lysate pulsed DCs respectively (Figure 5B).

A IFN-γ spots number

300

#

**

250 200 150


*

100 50 0

Treatment

B

Discussion

IFN-γ spots number

160 140

*

120

Control DC/Hepa DC/Hepa lysate

100 80 60 40 20 0

Treatment

Figure 5. DC-dependent antigen-specific CTL priming was impaired by co-incubation with lymphoma or hepatoma cells. DCs (1 × 106) were co-incubated with either 1 × 105 lymphoma or hepatoma cells. After co-incubation, DCs were pulsed with 1 × 105 tumor lysate for 4 hours and performed MLRs. DCs pulsed with either 1 × 105 tumor cell lysate or OVA as positive control. IFN-γ secretion by antigenic-specific CTL stimulating with either tumor cell lysate or SIINFEKL peptide in MLRs was determined by ELISPOT assay. Data were given as mean ± SEM, n = 6. These data were representative of three independent experiments, analyzed by Student’s t test. (A) DCs-dependent CTL priming was impaired by co-incubation with lymphoma cells. *p < 0.05 indicated for EG7 versus EG7 lysate; **p < 0.05 for EG7 versus OVA; #p < 0.001 for OVA versus control respectively. (B) DCs-dependent CTL priming was impaired by co-incubation with hepatoma cells. *p < 0.001 indicated for Hepa versus Hepa lysate. Hepa, Hepa 1-6.

secretion in MLRs was seriously impaired by tumor cells co-incubation. For example, EG7 cell pre-incubated imDCs could only produce 51 pg/ml IL-12 in the supernatants of MLRs, while EG7 lysate or OVA pulsed imDCs could produce 925 pg/ml and 3397 pg/ml, respectively (Figure 4A). In case of Hepa 1-6 co-incubated imDCs, the similar reduction of IL-12 can be observed (Figure 4B). We noticed that the controls in two independent experiments have different IL-12 levels. This might be due to the difference in MLRs performance, animal age or ELISA procedures. Co-incubation with tumor cells decreases CTL priming ability of DCs in vitro As tumor co-incubation decreased the DCs’ ability of T cell priming, additional experiments were needed to further explore if such co-incubation could impair the ability of antigenic-specific CTL priming which is essential for antitumor immunity. To this end, IFN-γ+ tumor antigenspecific CTL in MLRs was determined with ELISPOT assays Volume 5

It is well known that many tumors are potentially immunogenic (1). DCs are specialized APCs that recognize, acquire, process and present antigens to naïve T cells for the induction of an antigen-specific immune response (1). However, the presence of functional immunogenic mature DCs is rare in both human tumors, such as ovarian (3), breast tumors (4), prostate cancer (5) and in renal-cell carcinomas (6). In the present study, bone marrow-derived, GM-CSF and IL-4 induced myeloid imDCs were incubated in 8.0-12 μm TranswellTM system with lymphoma, hepatoma, and melanoma cells in vitro to directly observe the effects of co-incubated imDCs in surface molecule expression, IL-12 secretion, DCs-dependent T cell proliferation and tumor antigen-specific CTL priming. Our results showed that co-incubation with either TSA-lymphoma EG7 cells or TAA hepatoma and melanoma cells, decreased surface molecule expressions of CD54, CD11a, CD11b and MHC class II molecules on imDCs. It is reported that the onset and regulation of a specific immune response were resulted from immunological synapse (23, 24), which consists of three phases: contact acquisition, formation of an interaction plane and detachment of the T cell (24). In the first phase, the immunological-synapse formation occurs when moving T cells recognize DCs through peptide-MHC complexes and CD11a, CD11b, CD54, leading to a transient arrest in migration (25). Meanwhile, the impact of the type and activation status of the APCs on the duration of the cell-cell interaction seems to be correlated with the co-stimulatory capacity and activation potency of the APCs (26). Although several reports have described inhibitory effects of soluble tumor-derived factors such as IL-6, IL-10, TGF-β, vascular endothelial growth factor, and gangliosides on DCs (27-29), the down-regulation of surface molecules on imDCs induced by tumor cell co-incubation might also inhibit the direct interaction between T cell and DCs at the first phase of immunological synapse formation, hence decreasing the DC-dependent T cell activation and proliferation. However, in our experiments, we found some controversial results of a few surface markers on imDCs co-incubated with tumor cells from different origins. For example, CD40 was downregulated (p < 0.05) and CD86 was not altered (p > 0.05) on TSA-lymphoma incubated DCs, but these two molecules were up-regulated on hepatoma and melanoma incubated imDCs. These differences might be attributed to different

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types of tumor cells used for DCs co-incubation and their relevant antigenicity. Up-regulation of CD40 and CD86 on spleen DCs were also observed in animals after subcutaneous and intraperitoneal administration of lymphoma cells (11). For the mechanism of DC-T cell direct interaction, the fate of naïve Th cells is presumably determined by three signals that are provided by pathogen-primed mature DCs (30). Signal 1 is delivered through the T-cell receptor (TCR) when it engages an appropriate peptide-MHC complex. Signal 2 is referred to the fine balance of positive and negative co-stimulatory signals (30), which in collaboration with signal 1 induces immunity and is often measured as T cell expansion and differentiation into effector cells (31). Signal 3 comprises of signals delivered from the APCs to the T cells to commit their differentiation into Th1 cells, Th2 cells or CTLs (32). In the present study, the down-regulation of surface molecules on DCs was observed by tumor cell co-incubation. These results might suggest that tumor cell-incubated DCs could only generate minor signal 1 and signal 2 related molecules. The non-specific immunological synapse between naïve T cell and DC often generates a weak signal, such as minor tyrosine phosphorylation and calcium influx, which is not potent enough to initiate primary T-cell activation and proliferation (24). It was not surprising to find that tumor-treated DCs could achieve less DCs-dependent T cell proliferation and antigen-specific CTL priming. As IL-12 is one of the key mediators of signal 3 to promote Th1-cell or CTL development (33), the decrements of IL-12 secretion and effectors CTL priming in our data might indicate that tumor cells-incubated DCs failed to induce the differentiation of T cells with full function (34). As shown in our data, the present tumor cell-DC coincubation does reveal the DC dysfunction attributed to the direct interaction by tumor cells, thereby providing a method capable of dissecting the tumor cell-DC interaction in tumor microenvironment. However, we have to address that the stroma of tumor is much more complex than the present tumor cell and DC two co-culture. In fact, tumor tissues comprise of tumor cells, tumor-associated fibroblasts, vascular endothelial cells, extracellular matrix and different variety of immune cells, including DCs, macrophages, granulocytes and others. There are a complex interaction networks among these cells and elements. Cao’s group has demonstrated that regulatory DCs cell are generated after co-incubation with spleen stromal cells, which have inhibitory effect on T cell proliferation (16). Moreover, a number of molecules, such as cyclooxygenase-2 (COX2), prostaglandin E2 (PGE2), IL-10, IL-6, vascular endothelial growth factor (VEGF), gangliosides and TGF-β, secreted by tumor and tumor-associated macrophages, also suppress DC maturation and function (27-29). Therefore, the interaction among cell elements and their secretary molecules needs further extensive studies. Taken together, our results demonstrated that coincubation with tumor cells could down-regulate the surface molecules on imDCs and decrease the ability of DC to prime T cell proliferation. It was further revealed that the DCdependent antigen-specific CTL priming was also ablated, Volume 5

indicating that tumor-induced myeloid DC defects occurred in the present tumor cell-DC co-incubation condition. This co-culture system provides an experimental model in vitro for studies of immunological dysfunction in cancer, which is valuable for studying the tumor-immune cells direct interaction and for future immune-modulation drug screening.

Acknowledgements We thank Professor Xuetao Cao (Second Military Medical University, Shanghai, China) for kindly providing EG7 and EL4 cell lines. Also, we thank Mingxia Yan and Ming Yao for animal care. All authors concur with the submission and that the material submitted has not been previously reported and is not under consideration for publication elsewhere. We declare no competing financial interests.

References 1. Zou WP. Immunosuppressive networks in the tumor environment and their therapeutic relevance. Nat Rev Cancer. 2005;5:263-274. 2. Bergmann C, Strauss L, Zeidler R, Lang S, Whiteside TL. Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment. Cancer Immunol Immunother. 2007;56:1429-1442. 3. Zou WP, Machelon V, Coulomb-L’ Hermin A, et al. Stromalderived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nat Med. 2001;7: 1339-1346. 4. Iwamoto M, Shinohara H, Miyamoto A, et al. Prognostic value of tumor-infiltrating dendritic cells expressing CD83 in human breast carcinomas. Int J Cancer. 2003;104:92-97. 5. Troy A, Davidson P, Atkinson C, et al. Phenotypic characterisation of the dendritic cell infiltrate in prostate cancer. J Urol. 1998:160:214-219. 6. Troy AJ, Summers KL, Davidson PJT, et al. Minimal recruitment and activation of dendritic cells within renal cell carcinoma. Clin Cancer Res. 1998;4:585-593. 7. Gabrilovich D. Mechanisms and functional significance of tumor-induced dendritic-cell defects. Nat Rev Immunol. 2004;4: 941-952. 8. Orsini E, Guarini A, Chiaretti S, Mauro FR, Foa R. The circulating dendritic cell compartment in patients with chronic lymphocytic leukemia is severely defective and unable to stimulate an effective T-cell response. Cancer Res. 2003;63: 4497-4506. 9. Sharma S, Stolina M, Yang SC, et al. Tumor cyclooxygenase 2-dependent suppression of dendritic cell function. Clin Cancer Res. 2003;9:961-968. 10. Yang AS, Lattime EC. Tumor-induced interleukin 10 suppresses the ability of splenic dendritic cell to stimulate CD4 and CD8 T-cell responses. Cancer Res. 2003;63:2150-2157. 11. Sevko al, Barysik N, Perez L, Shurin MR, Gerein V. Differences in dendritic cell activation and distribution after intravenous, intraperitoneal, and subcutaneous injection of lymphoma cells in mice. Adv Exp Med Biol. 2007;601:257-264. 12. Moore MW, Carbone FR, Bevan MJ. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell. 1988;54:777-785. 13. Darlington GJ, Bernhard HP, Miller RA, Ruddle FH. Expression

Number 2

April 2008

140

14. 15.

16. 17.

18.

19. 20. 21. 22. 23.

24.

Dysfunction of Murine Dendritic Cells Induced by Incubation with Tumor Cells of liver phenotypes in cultured mouse hepatoma cells. J Natl Cancer Inst. 1980;64:809-819. Briles EB, Kornfeld. Isolation and metastatic properties of detachment variants of B16 melanoma cells. J Natl Cancer Inst. 1978;60:1217-1222. Whiteside TL, Stanson J, Shurin MR, Ferrone S. Antigenprocessing machinery in human dendritic cells: up-regulation by maturation and down-regulation by tumor cells. J Immunol. 2004;173:1526-1534. Zhang MH, Tang H, Guo ZH, et al. Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells. Nat Immunol. 2004;5:1124-1133. Berthier-Vergnes O, Gaucherand M, Peguet-Navarro J, et al. Human melanoma cells inhibit the earliest differentiation steps of human Langerhans cell precursors but failed to affect the functional maturation of epidermal Langerhans cells. Br J Cancer. 2001;85:1944-1951. Aicher A, Heeschen C, Mohanpt M, Cooke JP, Zeiher AM, Dimmeler S. Nicotine strongly activates dendritic cell-mediated adaptive immunity-potential role for progression of atherosclerotic lesions. Circulation. 2003;107:604-611. Gao FG, Wan DF, Gu JR. Ex vivo nicotine stimulation augents the efficacy of therapeutic bone marrow-derived dendritic cell vaccination. Clin Cancer Res. 2007:13:3706-3712. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245-252. Campoli M, Chang GC, Ferrone S. HLA-class I antigen loss, tumor immune escape and immune selection. Vaccine. 2002;20: 40-45. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 2003;3: 33-46. Gao FG, Khammanivong V, Liu WJ, Leggatt GR, Frazer IH, Fernando GJP. Antigen specific CD4+ T cell help is required to activate a memory CD8+ T cell to a fully functional tumor killer cell. Cancer Res. 2002;62:6438-6441. Friedl P, den Boer AT, Gunzer M. Tuning immune responses:

Volume 5

25. 26.

27. 28.

29. 30.

31. 32.

33.

34.

Number 2

diversity and adaptation of the immunological synapse. Nat Rev Immunol. 2005:5:532-545. Dustin ML, Bromley SK, Kan Z, Peterson DA, Unanue ER. Antigen receptor engagement delivers a stop signal to migrating T lymphocytes. Proc Natl Acad Sci U S A. 1997;94:3909-3913. Gunzer M, Weushaupt C, Hillmer A, et al. A spectrum of biophysical interaction modes between T cells and different antigen-presenting cells during priming in 3-D collagen and in vivo. Blood. 2004;104:2801-2809. Kiertscher SM, Luo J, Dubinett SM, Roth MD. Tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells. J Immunol. 2000;164:1269-1276. Shurin MR, Yurkovetsky ZR, Tourkova IL, Balkir L, Shurin GV. Inhibition of CD40 expression and CD40-mediated dendritic cell function by tumor-derived IL10. Int J Cancer. 2002;101: 61-68. Kao JY, Gong Y, Chen CM, Zheng QD, Chen JJ. Tumor-derived TGF-β reduces the efficacy of dendritic cell/tumor fusion vaccine. J Immunol. 2003;170:3806-3811. Young LJ, Wilson NS, Schnorrer P, et al. Dendritic cell preactivation impairs MHC calss II presentation of vaccines and endogenous viral antigens. Proc Natl Acad Sci U S A. 2007;104: 17753-17758. Kearney ER, Pape KA, Loh DY, Jenkins MK. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity. 1994;1:327-339. Curtsinger JM., Lins DC, Mescher MF. Signal 3 determines tolerance versus full activation of naïve CD8 T cells: dissociating proliferation and development of effector function. J Exp Med. 2003;197:1141-1151. Benvenuti F, Lagaudriere-Gesbert C, Grandjean I, et al. Dendritic cell maturation controls adhesion, synapse formation, and the duration of the interactions with naïve T lymphocytes. J Immunol. 2004;172:292-301. Hugues S, Fetler L, Bonifaz L, Helft J, Amblard F, Amigorena S. Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity. Nat Immunol. 2004;5:1235-1242.

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