Enhanced anti-cancer activity of human dendritic cells sensitized with gamma-irradiation-induced apoptotic colon cancer cells

Cancer Letters 335 (2013) 278–288

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Enhanced anti-cancer activity of human dendritic cells sensitized with gamma-irradiation-induced apoptotic colon cancer cells Sun Kyung Kim a, Cheol-Heui Yun b, Seung Hyun Han a,⇑ a

Department of Oral Microbiology and Immunology, DRI, and BK21 Program, School of Dentistry, Seoul National University, Seoul, Republic of Korea Animal Science and Biotechnology Major and World Class University Biomodulation Major, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 15 January 2013 Received in revised form 14 February 2013 Accepted 14 February 2013

Keywords: Dendritic cells Cancer cell-killing c-Irradiation Cytotoxic molecules HT-29

a b s t r a c t Properly sensitized dendritic cells (DCs) can be an effective immunotherapeutic against cancers. We investigated the phenotypic and functional changes in human DCs sensitized with c-irradiated colon cancer cell-line HT-29 (GIH). GIH induced maturation and activation of DCs. GIH-sensitized DCs showed increased cytotoxic activity against HT-29 through higher expression of perforin and granzyme B. They further induced expression of effector cytokines, cytotoxic molecules, and mucosal-homing receptor in autologous T-cells. Conclusively, these results suggest that effective anti-cancer activity is induced when DCs are sensitized with c-irradiated cancer cells via both direct augmentation of the cytotoxicity and indirect activation of T cells. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Dendritic cells (DCs) are the professional antigen-presenting cells that link innate and adaptive immunities and play an important role in the elimination of cancer cells. DCs sense, phagocytose, and digest malignant cells, and at the same time, migrate into draining lymph nodes to initiate the induction of adaptive immune responses [1]. In the lymph nodes, DCs present the epitopes derived from the cancer cells to naïve helper T (TH) cells, which in turn activate cytotoxic T lymphocytes (CTLs) to kill the cancer cells, mainly by releasing death-related molecules such as perforin and granzymes [2]. DCs also express cytokines and co-stimulatory molecules that affect the type, strength, and duration of adaptive immune responses [3] required for effective anti-cancer immune responses [1]. In addition to the role of DCs as antigen-presenting cells, DCs can also eliminate cancer cells directly. DCs provide cancer cells with death signals such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL), resulting in apoptotic death [4]. They also utilize soluble cytotoxic effectors like perforin and granzyme B to eliminate cancer cells [5]. Pre-clinical and clinical studies show convincing evidence on the activation of cellular immunity and cancer regression following ⇑ Corresponding author. Address: Department of Oral Microbiology and Immunology, DRI, and BK21 Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. Tel.: +82 2 740 8641; fax: +82 2 743 0311. E-mail address: [email protected] (S.H. Han). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.038

treatment of cancer with DCs. For example, vaccination with DCs carrying cancer antigens demonstrated improvement in the antigen-specific cytotoxic activity of CTLs against melanoma and reduction of the metastases [6]. In addition, patients with melanoma who received DCs pulsed with lysates of three allogenic melanoma cell lines elicited anti-melanoma immune responses, reduction of regulatory T cells (Treg), and prolonged survival [7]. For DC therapy, preparation of the cancer antigens to sensitize DCs is substantially important in maximizing efficacy [8]. Antigen-sensitizing methods commonly used in pre-clinical and clinical trials include: (i) transduction of viral vectors encoding tumor-associated antigen (TAA) peptides in DCs, resulting in a specific immune response limited to the transduced TAA [9]; (ii) fusion of DCs with cancer cells possessing multiple cancer antigens, but low fusion efficacy and immunosuppressive properties of the hybridoma obscure the efficacy of the therapy [10]; and (iii) sensitizing DCs with killed cancer cells that contain a number of various cancer-specific antigens [11]. Various methods have been introduced for the inactivation of cancer cells such as chemotherapeutic treatment, freeze-thawing, and ionizing radiation, which exhibit distinctive phenotypes and immunostimulating capacities in the activation of DCs [12]. Ionizing radiation, including c-rays, has been used as the conventional anti-cancer therapy for years because the local irradiation to the area of the tumor in the patient directly induces cancer cell death and limits progression of tumor [13]. Recently, c-irradiation was reported to induce immunogenic tumor cell death [12] and immunotherapy together with c-irradiation has

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focused on the development of cancer vaccines using DCs [14,15], calling for further studies into the mechanisms by which irradiated cancer cells modulate DCs and subsequent immune responses. In the present study, we investigated whether c-irradiated cancer cells could sensitize and enhance the anti-cancer immune properties of DCs targeting a human colon cancer cell-line, HT-29.

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30 min. Then, the cells were washed three times with cold PBS containing 2% FBS and analyzed by flow cytometry (FACSCalibur, BD Biosciences). At least 1  104 cells were acquired for each sample and dead cells and cell debris were gated out. All flow cytometric data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).

2.5. Intracellular staining of cytokines 2. Materials and methods 2.1. Reagents and chemicals Ficoll–Paque plus was obtained from GE Healthcare (Uppsala, Sweden). Antihuman CD14 magnetic particles were purchased from BD Biosciences (San Diego, CA, USA). Fetal bovine serum (FBS), penicillin–streptomycin solution, RPMI 1640 and DMEM were purchased from HyClone (Logan, UT, USA) and Trypsin–EDTA solution was purchased from Invitrogen (Carlsbad, CA, USA). Recombinant human GMCSF and interleukin (IL)-4 were purchased from Peprotech (Rocky Hill, NJ, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Annexin V-FITC apoptosis detection kit I for analyzing cancer cell death and 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE/CFSE) for fluorochrome-labeling cancer cells were purchased from BD Biosciences and Molecular Probes (Eugene, OR, USA), respectively. Monoclonal antibodies (mAbs) used for flow cytometric analyses were PE-labeled anti-human CD80 antibody (Ab), APC-labeled anti-human CD86 Ab, FITC-labeled anti-human HLA-DR, DP, DQ Ab for MHC class II, Alexa Fluor 647-labeled anti-human CD205 Ab, and PE-labeled anti-human IL-12p70 Ab were obtained from BD Biosciences. FITC-labeled anti-human TNF-a Ab, APC-labeled anti-human IL-6 Ab, APC-labeled anti-human IL-10 Ab, Alexa Fluor 647-labeled anti-human DC-SIGN Ab, APC-labeled anti-human PD-L1 Ab, PE-labeled antihuman PD-L2 Ab, PE-labeled or APC-labeled anti-human perforin Ab, Alexa Fluor 647-labeled anti-human granzyme B Ab, FITC-labeled or APC-labeled anti-human CD25 Ab, APC-labeled anti-human CD69 Ab, PE-labeled or APC-labeled anti-human CD3 Ab, PE-labeled anti-human CD4 Ab, PE-labeled anti-human CD8 Ab, Alexa Fluor 647-labeled anti-human FoxP3 Ab, APC-labeled anti-human a4b7, Alexa Fluor 647labeled CCR6 Ab and Alexa Fluor 647-labeled anti-human CCR9 Ab were purchased from BioLegend (San Diego, CA, USA). All isotype-matched control (IC) Abs were obtained from BD Bioscience or BioLegend. Concanamycin A and staurosporine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Z-AAD-CMK was obtained from Calbiochem (Darmstadt, Germany). 2.2. Generation of human monocyte-derived DC All experiments using human blood were conducted under the approval of the Institutional Review Board of the Seoul National University (IRB No. S-D20060001). Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized adult peripheral blood by density gradient centrifugation using Ficoll–Paque plus. Monocytes were isolated using anti-human CD14 magnetic beads (BD Biosciences). Then, 2  106 cells/ml of CD14+ cells were plated on 100-mm cell culture dishes and cultured in complete RPMI 1640 supplemented with 800 U/ml of GM-CSF and 500 U/ ml of IL-4 for 5 days. The cytokines were changed after 3 days. Expression of phenotypic markers of monocytes and iDCs were analyzed by flow cytometry (FACSCalibur, BD Biosciences). 2.3. Preparation of inactivated cancer cells and DC sensitization HT-29 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured on 100-mm cell culture dishes until 70–80% confluent in complete DMEM containing 10% FBS and 1% penicillin–streptomycin, and then detached by trypsinization using 0.25% trypsin–EDTA solution. Inactivated HT-29 cells were prepared and mixed with iDCs as follows. HT-29 cells were detached and re-suspended in complete DMEM at 1  106 cells/ml. To prepare c-irradiated HT-29 cells (GIH), the cells were irradiated with c-rays at 10, 50, or 100 Gy. Freeze-thawed HT-29 (FTH) cells were snap-frozen in liquid nitrogen, and immediately thawed in a 37 °C water bath, and this procedure was repeated three times. To prepare chemotherapeutic-induced apoptotic HT-29 (SH) cells, the cells were treated with 5 nM of staurosporine for 48 h. Gamma-irradiation/staurosporine-co-treated HT-29 (ISH) cells were also prepared to examine the combined effect of radiation and chemotherapy. The condition of the cells was analyzed by propidium iodide (PI) and annexin V-FITC staining according to the manufacturer’s instructions followed by flow cytometric analyses. Live HT-29 cells (LH) were used as a negative control. For sensitization, the iDCs were incubated with inactivated HT29 cells for 24–48 h. 2.4. Characterization of DCs iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 cells (1  106 cells/ml) in the presence of GM-CSF and IL-4 for 48 h. DCs were harvested and stained with fluorochrome-conjugated mAbs specific to typical DC markers including CD80, CD86, MHC class II, CD205, PD-L1 and PD-L2 on ice for

iDCs (1  106 cells/ml) were stimulated with either live or killed HT-29 cells (1  106 cells/ml) for 24 h in the presence of GM-CSF and IL-4. Then, brefeldin A (10 lg/ml) was added to the culture at 8 h before the harvest. The cells were stained with mAbs specific to MHC class II or DC-SIGN, which HT-29 cells do not express. The cells were fixed with 4% paraformaldehyde for 15 min, and then permeabilized with PBS containing 0.1% saponin for another 15 min on ice. The permeabilized cells were stained for human IL-6, TNF-a, IL-12p70, or IL-10 on ice for 30 min, and then washed three times with cold PBS containing 0.1% saponin. The cells were acquired and analyzed as described above.

2.6. Phagocytosis assay To label HT-29 cells, the cells (5  106 cells/ml) were suspended in PBS containing 10 lM of CFSE for 15 min at 37 °C. The cells were washed with PBS three times, re-suspended in complete DMEM at 1  106 cells/ml, and then subsequently irradiated (50 Gy). The irradiated CFSE-HT-29 cells were incubated for 48 h, and mixed with iDCs (1  106 cells/ml) at various culture ratios (DC:HT-29 = 1:0.1, 1:0.2, 1:0.5, 1:1, and 1:2) for 12 h at 37 °C or 4 °C to measure specific uptake and non-specific uptake, respectively. Then, DC uptake was analyzed by flow cytometry.

2.7. Activation of autologous T cells by DCs sensitized with GIH DCs sensitized with GIH (1  105 cells) and autologous CD14+ cells-depleted PBMCs (1  106 cells) were plated in flat bottom 96-well cell culture plates and incubated for 4 days. Anti-human CD3 Ab was used with either anti-human CD4 Ab or CD8 Ab to analyze the CD4+ or CD8+ T cell population. To analyze Treg, the cells were stained with anti-human CD4 Ab and anti-human CD25 Ab, and then intracellular expression of FoxP3 was analyzed using anti-human FoxP3 Ab. To examine T-cell activation, staining with anti-human CD25 Ab and CD69 Ab followed by flow cytometric examination was performed. To determine the expression of gut mucosal-homing receptor on T cell subpopulations, a mAb specific to human a4b7, CCR6, or CCR9 was used. To analyze expression of cytokines in CD4+ cells and CD8+ T cells, phorbol 12-myristate 13-acetate (PMA, 0.5 lg/ml) and ionomycin (0.5 lg/ ml) were added to the DC-PBMC cultures 6 h before harvest. Brefeldin A (10 lg/ ml) was also added for 6 h to block the release of cytokines. After surface staining with either anti-human CD4 Ab or CD8 Ab, the cells were fixed, permeabilized, and stained with anti-human mAb specific to IFN-c, IL-17A, IL-4, granzyme B, or perforin. The cells were analyzed by flow cytometry.

2.8. Measurement of cancer cell-killing activity of DCs (JAM test) Live HT-29 cells (1  105 cells/ml) were labeled with 1 lCi/ml of [3H]-thymidine for 12 h at 37 °C. The cells were re-suspended in RPMI 1640 and seeded in 96-well flat bottom plates at 1  104 cells/well. DCs sensitized with GIH were mixed with the target cells at an effector to target ratio of 20:1 as described previously [5] and incubated for 48 h. The cells were harvested and radioactivity was measured. The cytotoxicity (cancer cell-killing activity) of the DCs was calculated with the following formula: cpm [1-{(target + killer)/target alone}]  100.

3. Results 3.1. GIH efficiently induces maturation of DCs First, we characterized the cell death in GIH. Annexin V- and PIpositive apoptotic cell populations increased as the irradiation intensity increased (Fig. 1A) and as the incubation time increased (Fig. 1B). Next, iDCs were treated with the irradiated HT-29 cells and DC maturation was analyzed. HT-29 cells undergoing apoptosis induced expression of co-stimulatory molecules (CD80 and CD86) and MHC class II on iDCs in dose- and time-dependent manner (Fig. 1C and D). In addition, iDCs phagocytosed the irradiated HT-29 cells (Fig. 1E). These results suggest that irradiated, apoptotic HT-29 cells induce the activation and maturation of DCs.

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A

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Radiation intensity (at 48 h) 10 Gy

50 Gy

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CD80

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E _

1:0.1

1:0.2

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1:2

DC-S SIGN

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4C 4ºC

CFSE-GIH Fig. 1. GIH efficiently enhances DC maturation. HT-29 cells (1  106 cells/ml) were (A) irradiated with c-rays at various intensities (0, 10, 50, and 100 Gy) followed by incubation for 48 h, or (B) exposed to c-rays (50 Gy) followed by incubation for 0, 24, 48, or 72 h. Then, the death of the HT-29 cells was analyzed by flow cytometry after PI and annexin V staining. Numbers indicate the percentage of each cell population. The GIH (1  106 cells/ml) prepared with (C) certain radiation intensities for 48 h or (D) 50 Gy for certain incubation time periods were co-cultured with iDCs (1  106 cells/ml) for 48 h, and then expression of DC maturation markers was analyzed. Numbers in each histogram indicate the mean fluorescence intensities (MFI). (E) iDCs (1  106 cells/ml) were cultured with CFSE-stained GIH at various culture ratios (DC:HT-29 = 1:0.1, 1:0.2, 1:0.5, 1:1 and 1:2) for 12 h at 37 °C or 4 °C, and then the uptake of the GIH by DC was analyzed by flow cytometry. Numbers indicate the percentage of CFSE-positive DCs. One of three similar results is shown.

3.2. GIH and ISH activated DCs more potently than FTH or SH We comparatively examined the DC-activating abilities of freezethawed (FTH), staurosporine-treated (SH), or irradiated/staurosporine-treated (ISH) HT-29 cells. We chose a staurosporine concentration that induced a similar degree of cell death to that of GIH (data not shown). When iDCs were stimulated with live HT-29 cells (LH), GIH, FTH, SH, and ISH for 48 h, GIH and ISH significantly up-regulated the expression of CD80, CD86, MHC class II and CD205. At the same time, GIH and ISH remarkably decreased the expression of the programmed cell death ligands, PD-L1 and PD-L2 (Fig. 2B), which are known to negatively regulate T cells [16]. The

GIH and ISH did not show significant induction of cytokines, while FTH showed an increase in the expression of IL-12p70 and IL-10 (Fig. 2C). Phenotypic changes and cytokine expression were hardly observed in DCs treated with SH. These results indicate that c-irradiation of HT-29 cells was more potent at inducing the maturation and activation of DCs than other inactivation treatments in HT-29 cells. 3.3. GIH potentiates cytotoxicity of DCs partially through perforin and granzyme B Next, we evaluated the cancer cell-killing activity of DCs sensitized with the inactivated HT-29 cells. The DCs (effectors) were

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TNF-alpha (% of positive cells)

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PD-L2

Fig. 2. GIH treatment induces DC maturation more potently than other inactivation treatments in HT-29 cells. (A) HT-29 cells were treated with c-irradiation (50 Gy) with or without staurosporine (5 nM), freeze-thawing (3 cycles), or staurosporine (5 nM), and then the cells were incubated for 48 h. Cell death was analyzed by flow cytometry after PI and annexin V staining. (B) iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 (1  106 cells/ml) for 48 h, and then the expression of maturation markers on DCs was analyzed by flow cytometry. MFI are presented in each histogram. (C) To analyze cytokine production by DCs, iDCs were stimulated with either live or inactivated HT-29 cells for 24 h. Brefeldin A (10 lg/ml) was added 8 h before harvest. Intracellular expression of each cytokine was analyzed. LH, GIH, FTH, SH, and ISH indicate live HT-29, c-irradiated HT-29, freeze-thawed HT-29, staurosporine-treated HT-29, and c-irradiation/staurosporine-co-treated HT-29, respectively.

mixed with radio-labeled live HT-29 (targets) and the killing activity was examined by measuring DNA fragmentation [17]. GIH and ISH produced significantly higher cytotoxicity than FTH or SH

(Fig. 3A). It has been suggested that DCs can exhibit enhanced cancer cell-killing activity by up-regulating the expression of cytotoxic molecules such as perforin and granzyme B [5]. Notably, the

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Cyttotoxicity (%)

A

DC : HT HT-29 29 = 20 20:1 1

B

+ Live HT-29

12 h

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LH

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5.0

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22.7

16.0

12.9

21.2

7.7

6.5

21.1

11.4

8.6

20.6

6.9

3.2

23.4

17.8

7.2

24.2

0h

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0h

DC sensitized with

DC sensitized with

24 h

48 h

NT

LH

GIH

FTH

SH

ISH

4.9

5.9

21.6

8.8

23.8

24.3

33.2

25.4

44.4

40.1

36.9

52.7

13.8

15.1

46.3

27.2

20.6

47.8

23.6

12.4

46.4

44.1

27.4

48.0

Granzyme B

Perforin

C 40

30

*

20

*

10

0 Concanamycin A

Cytottoxicity y (%)

Cytottoxicity y (%)

40

30

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0 0 nM

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Z-AAD-CMK

0 nM

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Fig. 3. GIH potentiates DC cancer cell-killing activity mediated through perforin and granzyme B. (A) iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 cells (1  106 cells/ml) for 48 h. Then, the cells were harvested and co-cultured with [3H]-thymidine-labeled live HT-29 at an effector to target cell ratio of 20:1 for another 48 h. The cancer cell-killing activity of DCs was measured by determination of the radioactivity. (B) iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 cells (1  106 cells/m) for 48 h. Then, the DCs were co-cultured with live HT-29 cells for various time periods (0, 12, 24, and 48 h) and the expression of perforin and granzyme B in DC was analyzed by flow cytometry. The numbers on each panel indicate the percentage of perforin- and/or granzyme B-expressing cells. (C) GIHDCs were co-cultured with radio-labeled live HT-29 cells (effector:target = 20:1) in the presence of concanamycin A (1 and 2 nM) or Z-AAD-CMK (1 and 2 nM) for 48 h, and then the cytotoxicity of the DCs was analyzed as described in materials and methods. One of three similar results is shown.

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A

Gate: CD4+ cells DC : autologous PBMC = 1:10 No DC

iDC

LH-DC LH DC

GIH-DC GIH DC

FTH-DC FTH DC

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ISH-DC ISH DC % of (+) pop. MFI of (+) pop.

CD25

CD69

B

Gate: CD8+ cells DC : autologous PBMC = 1:10 No DC

iDC

LH-DC

GIH-DC

FTH-DC

SH-DC

ISH-DC % of (+) pop pop. MFI of (+) pop.

CD25

CD69

DC : autologous t l PBMC = 1:10 1 10

C iDC

LH-DC

GIH-DC

FTH-DC

SH-DC

ISH-DC

C CD3

_

CD25 5

CD4

C CD3

Foxp3

CD8 Fig. 4. GIH-DCs induce preferential proliferation and activation of TH cells. iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 cells (1  106 cells/ ml) for 48 h, and then the cells were harvested and incubated with autologous PBMCs (1  107 cells/ml). Expression of CD25 and CD69 on (A) CD4+ T cells and (B) CD8+ T cells was analyzed at 48 h. Percentage and MFI of CD69+ or CD25+ T cells are presented in the upper right on each histogram panel. (C) Changes in the ratios of TH cells (CD3+ CD4+ cells), Treg (CD4+ CD25+ Foxp3+ cells), and CTL (CD3+ CD8+ cells) among the PBMCs were analyzed by flow cytometry. The number on the upper right of each panel shows the percentage of the T cell subpopulation. One of three similar results is shown.

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PMA+Io

A

W/O PMA+Io 0.8

iDC

LH-DC

GIH-DC

FTH-DC

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ISH-DC

8.9

8.8

13.9

7.9

9.8

14.1

0.7

0.1

1.6

0.7

0.5

1.2

0.6

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IL-4 PMA+Io

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iDC

LH-DC

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14.1

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24.8

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IL-17A

0.7

IL-4 Fig. 5. T cells increase IFN-c and IL-17A production in response to GIH-DCs. (A and B) iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 for 24 h, and then, the cells were co-cultured with autologous PBMCs (1  107 cells/ml) for 4 days. Before harvest, the cells were stimulated with PMA (0.5 lg/ml) and ionomycin (0.5 lg/ml) and treated with brefeldin A (10 lg/ml) for 6 h. The cells were stained with anti-human CD4 and CD8 mAb and intracellular expression of IFN-c, IL-17A and IL-4 was analyzed by flow cytometry. The number in each panel indicates the percentage of IFN-c, IL-17A, or IL-4-producing cells out of CD4-positive or CD8-positive cells. One of three similar results is shown.

GIH-treated DCs (GIH-DCs) produced an increase in the intracellular expression of perforin and granzyme B in a time-dependent manner during co-culture with target cells (Fig. 3B). Coincidently, addition of concanamycin A (perforin inhibitor) or Z-AAD-CMK

(granzyme B inhibitor) to the co-culture significantly attenuated the killing activity of the DCs (Fig. 3C). These results indicate that GIH stimulates DCs to kill the cancer cells and perforin and granzyme B are, at least in part, involved in this effect.

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A

Gate: CD4+ cells No DC

iDC

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FTH-DC

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ISH-DC

12.8%

6.4%

9.7%

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% of (+) pop.

897

926

941

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Granzyme B 2.3%

1.3%

39

41

0.3%

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44

45

45

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Perforin

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Gate: CD8+ cells No DC 47.9% 748

iDC

LH-DC

49.2% 635

44.5% 630

GIH-DC 50.8% 813

FTH-DC 43.8% 721

SH-DC 44.9% 719

ISH-DC 49.6% 783

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Granzyme B 22.9%

16.5%

31

30

10.2% 29

18.0%

14.9%

16.4%

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30

30

30

32

Perforin Fig. 6. GIH-DCs enhance the intracellular expression of cytotoxic molecules in T cells. (A and B) iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 cells for 24 h followed by co-culture with autologous PBMCs (1  107 cells/ml) for an additional 4 days. The cells were harvested and stained with anti-human CD4 and CD8 mAb and intracellular expression of perforin and granzyme B in each T cell subpopulation was analyzed by flow cytometry. The number in each panel indicates the percentage and MFI of perforin or granzyme B-expressing cells out of CD4+ or CD8+ cells. One of three similar results is shown.

3.4. GIH-DCs induce T cell activation Since DCs can kill cancer cells indirectly by activating T cells [18], we next investigated T-cell activation and differentiation induced by the GIH-DCs. When GIH-DCs or ISH-DCs were cocultured with the CD14-depleted autologous PBMCs, the expression of T-cell activation markers (CD25 and CD69) was augmented on both CD4+ and CD8+ T cells (Fig. 4A and B). Under the same condition, the GIH-DCs and ISH-DCs increased the CD4+ T cell population, whereas there was a minimal change in the proportion of Treg or CD8+ T cells (Fig. 4C).

3.5. GIH-DCs favor TH1-type activation DCs can modulate TH1/TH2 polarization, preferentially activating cell-mediated and humoral immunity, respectively [19]. Therefore, we examined T-cell polarization induced by DCs sensitized with the aforementioned HT-29 cells. The DCs were co-cultured with autologous PBMCs for 4 days followed by stimulation with PMA and ionomycin for 6 h to analyze the production of IFN-c,

IL-4 and IL-17A. GIH-DCs and ISH-DCs induced higher expression of IFN-c and IL-17A in CD4+ cells than FTH-DCs or SH-DCs, but they induced little IL-4 (Fig. 5A). In CD8+ T cells, GIH-DCs and ISH-DCs together with SH-DCs induced IFN-c expression but not IL-17A or IL-4 (Fig. 5B).

3.6. GIH-sensitized DCs augment the expression of perforin and granzyme B in T cells To validate whether the cytotoxic ability of T cells was improved by the DCs, we subsequently examined the expression of perforin and granzyme B in CD4+ and CD8+ T cells. When the autologous PBMCs were co-cultured with GIH-DCs and ISH-DCs, there was not much change in the expression of granzyme B and a slight increase in perforin in CD4+ cells. Also, CD4+ T cells cultured with LH-DCs or SH-DCs showed an increase in expression of granzyme B, but not perforin, compared with CD4+ T cells cultured with iDC (Fig. 6A). Likewise, CD8+ T cells exhibited higher expression of granzyme B in response to GIH-DCs and ISH-DCs than the cells stimulated with iDCs, LH-DCs, FTH-DCs, or SH-DCs (Fig. 6B).

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S.K. Kim et al. / Cancer Letters 335 (2013) 278–288

A

Gate: CD4+ cells No DC

iDC

LH-DC

24.9%

12.5%

16.8%

119

79

89

GIH-DC 33.3% 164

FTH-DC

SH-DC

ISH-DC

34.2%

22.1%

28.9%

% of (+) pop.

153

110

127

MFI of (+) pop.

α4β7 16.0%

18.8%

19.5%

20.5%

20.3%

22.2%

19.6%

43

46

44

47

47

48

48

CCR6 2.1%

6.9%

8.0%

5.2%

7.7%

6.5%

4.8%

67

121

153

175

147

172

139

CCR9

B

Gate: CD8+ cells

No DC

iDC

LH-DC

49.2%

43.0%

38.7%

49

48

47

GIH-DC 54.1% 59

FTH-DC

SH-DC

ISH-DC

50.7%

47.9%

50.7%

% of (+) pop.

52

51

55

MFI of (+) pop.

α4β7 13.8% 37

13.9%

11.4%

13.7%

13.4%

13.5%

13.5%

44

40

46

50

45

43

CCR6 3.9%

3.9%

3.4%

2.5%

3.1%

3.0%

2.4%

120

121

111

140

98

119

127

CCR9 Fig. 7. GIH-DCs augment gut mucosal-homing receptor, a4b7 integrin. (A and B) iDCs (1  106 cells/ml) were stimulated with either live or inactivated HT-29 for 24 h, and then the cells were co-cultured with autologous PBMCs (1  107 cells/ml) for an additional 4 days. The cells were harvested and stained with anti-human CD4 and CD8 mAb and the expression of gut-homing integrin (a4b7) and CC chemokine receptors (CCR6 and CCR9) on CD4+ T and CD8+ T cells was analyzed by flow cytometry. The number in each panel indicates the percentage and MFI of a4b7, CCR6, or CCR9-expressing cells. One of three similar results is shown.

3.7. GIH-sensitized DCs enhance gut mucosal-homing receptor, a4b7, on T cells DCs regulate differentiation, proliferation, activation, and even expression of homing properties of T cells [20]. We finally investi-

gated whether the GIH-DCs could modulate the expression of homing receptors on the T cells. Autologous PBMCs were cultured with the GIH-DCs, FTH-DCs, SH-DCs, or ISH-DCs and the expression of a4b7, CCR6 or CCR9 on CD4+ and CD8+ T cells was analyzed. All GIH-DCs, FTH-DCs, SH-DCs, and ISH-DCs augmented the

S.K. Kim et al. / Cancer Letters 335 (2013) 278–288

expression of a4b7 on CD4+ T cells (Fig. 7A). Likewise, CD8+ T cells showed a substantial increase in the expression of a4b7 when the cells were co-cultured with GIH-DCs and ISH-DCs (Fig. 7B). On the other hand, all stimuli increased CCR9 expression on CD4+ T cells with marginal change in the CCR6 expression (Fig. 7A). Under the same condition, a minimal change was observed in the expression of CCR6 and CCR9 on CD8+ T cells (Fig. 7B).

4. Discussion Chemotherapy and radiotherapy are widely used treatments for cancer, but their adverse effects are often inevitable. Recently, immunotherapy using DCs has been considered as an alternative and promising cancer treatment. Consequently, the challenge in this area is preparing effective DCs. Here, we demonstrate that c-irradiation-induced apoptotic cancer cells could effectively stimulate human DCs to induce anti-cancer immune responses. Our results show that c-irradiation is superior to other inactivation methods such as freeze-thawing or staurosporine treatment. Remarkably, GIH effectively sensitized DCs leading to augmentation of cancer cell-killing activity, not only directly by increasing the expression of perforin and granzyme B, but also indirectly by activating T-cells. Our findings suggest that c-irradiation of cancer cells enhances the immunogenicity of DCs, which are congruent with the in vivo result that local irradiation to tumor-bearing microenvironment not only induces death of cancer cells, but also activates immune cells surrounding the tumor [21]. Previous studies have demonstrated that irradiated cancer cells provide ‘‘find and kill me’’ signals to various immune cells including DCs and CD8+ T cells by exposing calreticulins and/or increasing the expression of MHC class I and adhesion molecules [22,23]. Concomitantly, release of alarming molecules such as high mobility group box-1, heat shock proteins and nucleic acids from dead cancer cells can activate various toll-like receptors (TLRs), including TLR4, TLR7, and TLR9 of DCs, and mediate inflammatory responses by producing IL-12 and type I IFNs, respectively [24,25]. Moreover, ionizing radiation makes cancer cells sensitive to death signals from cytotoxic effector proteins such as TRAIL and FasL [26]. Upon exposure to GIH, DCs directly exert cytotoxicity against HT-29 cells. The increased cytotoxicity of DCs has also been reported to occur when DCs are stimulated with LPS or IFN-c [27]. An accumulating number of studies have demonstrated that DCs utilize cytotoxic soluble molecules such as perforin and granzymes related to pore-forming and granule-mediated apoptosis together with death receptors such as TRAIL and FasL [4,5]. Our findings suggest that perforin and granzyme B might be involved in GIHsensitized DCs since GIH increases the expression of those effector molecules and blocking of these molecules attenuates, at least partially, the cytotoxic ability of DCs. However, death receptormediated apoptosis does not seem to be associated with cancer cell-killing since the GIH could not induce the expression of TRAIL or FasL in DCs, while FTH dramatically increased those molecules (data not shown). Remarkably, colon cancer cells are known to be insensitive to TRAIL or FasL, unlike other types of cancer cells [28,29]. For this reason, the FTH-DCs showed no increase in the cancer cell-killing activity even though the cells expressed higher levels of TRAIL and FasL than GIH-DCs. Aside from an increase in the cancer cell-killing activity, GIHDCs are likely to stimulate the cell-mediated immunity that might additionally contribute to anti-cancer immunity for the following reasons. First, GIH-DCs sustained a higher CD4+ T cell proportion than other DCs. In this regard, the higher MHC class II and lower PD-L1 expression on the GIH-DCs could be responsible for the specific increase and/or survival of the CD4+ T cell population in the

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view of the fact that blockade of MHC class II interfered with T cell proliferation [30] and interaction between PD-1 and PD-L1 was negatively correlated with CD4+ T cell expansion and activation [16]. Second, GIH-DCs activated both CD4+ and CD8+ T cells to express IFN-c. In particular, induction of IFN-c expression might contribute to anti-cancer activity in light of the fact that IFN-c suppresses the invasive growth of cancer cells [31], and enhances sensitivity of cancer cells to death ligand-mediated death and antigen presentation by increasing MHC proteins [32,33]. Third, GIHDCs induced the expression of cytotoxic molecules, granzyme B and perforin, in CD8+ T cells. Granzyme B and perforin are important markers for the cytotoxicity of CD8+ T cells in immunotherapy [34]. Finally, GIH-DCs stimulated T cells to express the homing receptors a4b7, implying that GIH-DCs help with trafficking and retention of T cells to the gut mucosa by increasing the expression of gut mucosal-homing integrin, a4b7. The results of the present study suggest that GIH and GIH-DCs are promising immunotherapeutics. However, our study has limitations that must be addressed in further studies. First, the DCs used in the present study were derived from normal subjects, but not from patients with cancer. Since DCs from patients with cancer often show tolerance to immuno-stimulants and are less responsive to antigens [35], it is uncertain if DCs from patients with cancer are as responsive to c-irradiated cancer cells as DCs from normal subjects. Second, various types of cancer cells isolated from patients with cancer should be utilized. Despite these limitations, the present study potentially widens the knowledge of phenotypic and functional changes of DCs in response to c-irradiation-induced apoptotic cancer cells. In conclusion, our findings indicate that the c-irradiation of cancer cells is a promising method for cancer antigen preparation leading to effective DC-mediated anti-cancer immune responses. Conflict of interest statement None declared. Acknowledgments This work was supported by grants from the Expansion of Nuclear R&D Infrastructure Program through the Korea Science and Engineering Foundation (2008-01571), the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (2012-0008693 and 2012-0000492), and the R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. References [1] R.M. Steinman, J. Banchereau, Taking dendritic cells into medicine, Nature 449 (2007) 419–426. [2] K.L. Knutson, M.L. Disis, Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer immunology, immunotherapy, Cancer Immunol. Immunother. 54 (2005) 721–728. [3] J. Banchereau, R.M. Steinman, Dendritic cells and the control of immunity, Nature 392 (1998) 245–252. [4] G. Lu, B.M. Janjic, J. Janjic, T.L. Whiteside, W.J. Storkus, N.L. Vujanovic, Innate direct anticancer effector function of human immature dendritic cells. II. Role of TNF, lymphotoxin-alpha(1)beta(2), Fas ligand, and TNF-related apoptosisinducing ligand, J. Immunol. 168 (2002) 1831–1839. [5] G. Stary, C. Bangert, M. Tauber, R. Strohal, T. Kopp, G. Stingl, Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells, J. Exp. Med. 204 (2007) 1441–1451. [6] J. Steitz, J. Bruck, J. Knop, T. Tuting, Adenovirus-transduced dendritic cells stimulate cellular immunity to melanoma via a CD4(+) T cell-dependent mechanism, Gene Ther. 8 (2001) 1255–1263. [7] M.N. Lopez, C. Pereda, G. Segal, L. Munoz, R. Aguilera, F.E. Gonzalez, A. Escobar, A. Ginesta, D. Reyes, R. Gonzalez, A. Mendoza-Naranjo, M. Larrondo, A. Compan, C. Ferrada, F. Salazar-Onfray, Prolonged survival of dendritic cellvaccinated melanoma patients correlates with tumor-specific delayed type IV

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