In vitro anti-tumor immune response induced by dendritic cells transfected with EBV-LMP2 recombinant adenovirus

BBRC Biochemical and Biophysical Research Communications 347 (2006) 551–557 www.elsevier.com/locate/ybbrc

In vitro anti-tumor immune response induced by dendritic cells transfected with EBV-LMP2 recombinant adenovirus Ying Pan a, Jinkun Zhang a

a,*

, Ling Zhou b, Jianmin Zuo b, Yi Zeng

b

Department of Onco-pathology and the Key Immunopathology Laboratory of Guangdong Province, Shantou University Medical College, Shantou 515041, China b Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China Received 12 May 2006 Available online 27 June 2006

Abstract Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is a high-incidence tumor in southern China. Latent membrane proteins 2 (LMP2) is a subdominant antigen of EBV. The present study was to develop a dendritic cells (DCs)-based cancer vaccine (rAd-LMP2-DC) and to study its biological characteristics and its immune functions. Our results showed that LMP2 gene transfer did not alter the typical morphology of mature DC, and the representative phenotypes of mature DC (CD80, CD83, and CD86) were highly expressed in rAd-LMP2-DCs. The expression of LMP2 in rAd-LPM2-DCs was about 84.54%, which suggested efficient gene transfer. Transfected DCs markedly increased antigen-specific T-cell proliferation. The specific cytotoxicity against NPC cell was significantly higher than that in controls (p < 0.05), and enhanced with increased stimulations by transfected DCs. In addition, phenotypic analysis demonstrated that the LMP2-specific CTLs consisted of both CD4+ and CD8+ T cells. These results showed that development of DC-based vaccine by transfection with malignancy-associated virus antigens could elicit potent CTL response and provide a potential strategy of immunotherapy for EBV-associated NPC.  2006 Elsevier Inc. All rights reserved. Keywords: Dendritic cell; Nasopharyngeal carcinoma; Epstein-Barr virus; Gene transfer; Cytotoxic T lymphocyte; Cancer vaccine

Cytotoxic T lymphocytes (CTLs) recognize peptides derived from the intracellular breakdown of foreign antigens and present these peptides at the cell surface as a complex with major histocompatibility complex (MHC) class I molecules. Such CTLs play an important role in controlling virus infection. The virus-induced CTL response tends to focus on a few immunodominant peptide epitopes whose identities are specific for the particular MHC type of the host. This study concerns the CTL response to EpsteinBarr virus (EBV), a herpesvirus commonly associated with nasopharyngeal carcinoma (NPC). Among the EBV-associated NPC patients, the proteins of EBV expressed on tumor cells are very limited, only latent class II EBV anti-

*

Corresponding author. Fax: +86 754 8557562. E-mail address: [email protected] (J. Zhang).

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.214

gens such as the latent EBV nuclear antigens (EBNA1) and latent membrane proteins (LMP1 and LMP2) can be detected on NPC cells. Many human leukocyte antigen (HLA) class I restricted epitopes of LMP2 have been identified and their sequences are conserved, and LMP2 is thus the most frequently recognized protein by CTLs [1]. LMP2 constitutes potentially the major target antigen for immunotherapy of NPC. In our study, we sought to develop an efficient protocol to induce a strong LMP2-specific CTL response against tumor cells of NPC. Dendritic cells (DCs) are highly efficient and specialized antigen-presenting cells (APC) that are the only ones that can stimulate the native T cell and activate antigen-specific CTLs [2,3]. In vivo, immature DCs develop from hematopoietic progenitors and are located strategically at body surfaces, where they play a sentinel role in capturing and processing antigens.

552

Y. Pan et al. / Biochemical and Biophysical Research Communications 347 (2006) 551–557

Following antigen exposure, DCs migrate to lymphoid organs and acquire potent antigen-presenting function. Mature DCs process antigens efficiently by both MHC class I and II pathways with upregulation of cell surface adhesion molecules such as CD54 (ICAM1) and of costimulatory molecules such as CD80 and CD86. DCs have demonstrated potent anti-tumor properties in a variety of experimental models [4–6]. Recently some reports showed that calcium-signaling agents could induce maturation of DCs derived from peripheral blood monocytes [7,8]. DCs activated with calcium-signaling agents, in the presence of cytokines in serumfree medium, rapidly express mature DC marker, CD83, and high levels of co-stimulatory molecules within 96 h of culture. These activated DCs can efficiently sensitize T cells to recognize tumor cells through tumor antigens expressed by tumor cells. In our study, we prepared DCs by adenoviral transfection with EBV-LPM2 and calcium ionophore treatment. The acquired DC vaccine could stimulate T cells and elicit the potent antigen-specific CTLs activity against NPC cells. Materials and methods Nasopharyngeal carcinoma cell culture. Nasopharyngeal carcinoma cell line (CNE-2) which contains LMP2 gene [9] was obtained from the Institute for Viral Disease Control and Prevention of the Chinese Center for Disease Control and Prevention (Chinese CDC). The CNE-2 cells were grown in complete RPMI medium 1640 (Gibco, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS, Hyclone), 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin. Preparation of DCs. Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors by FicollHypaque (d = 1.077 g/ml) density-gradient centrifugation. Such PBMCs were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. After incubation for 2 h at 37 C in 5%CO2, the nonadherent cells were removed. The adherent cells as monocytes were harvested and resuspended in macrophage serum-free medium (M/-SFM; Gibco). The monocytes were then plated in a 24-well tissue-culture plate (Costar, USA) at 2.5 · 106 cells/well supplemented with 50 ng/ml rhGM-CSF (Peprotech, USA). This combination of M/-SFM and rhGM-CSF, which constitutes basal culture medium for all monocytes and DCs in this study, is henceforth referred to in the text simply as SFM/G. The monocytes were cultured for 24–48 h at 37 C in 5%CO2. To obtain mature DCs, the cells were treated with calcium ionophore A23187 (Sigma) at a concentration of 150 ng/ml for additional 48 h. The mature DCs were then collected and were analyzed for DC typical phenotypes by fluorescence-activated cell sorter (FACS) analysis or co-cultured with T cells for sensitization assays. Preparation of adenovirus transfected DCs. Recombinant serotype 5 adenoviruses encoding the LMP2 gene (rAd-LMP2) were obtained from the Institute for Viral Disease Control and Prevention of the Chinese CDC. The virus stocks were proliferated in human embryonic kidney (293) cells in DMEM (Gibco) supplemented with 2% heat-inactivated FCS and purified through cesium chloride (Sigma) gradient ultracentrifugations [10]. Viral particle concentration was determined by UV absorbance at 260 nm [11], and final viral titers were 1011 plaque-forming units (pfu). The monocytes were cultured in SFM/G for 48 h as described previously. The cells were harvested as immature DCs and were resuspended at 1 · 106 cells/200 ll in serum-free medium. The recombinant adenoviruses encoding the LMP2 gene were then added to infect immature DCs at multiplicities of infection (MOI) 200. Infection was allowed to proceed for 2 h at 37 C. Then fresh SFM/G was added to bring the cultures to 2 ml per well. One hour after adenoviruses transfection of immature DCs,

calcium ionophore A23187 was added at a concentration of 150 ng/ml. Transfected cells were cultured for additional 48 h and the mature rAdLMP2-DCs were harvested. To determine the viability of adenovirusesinfected DCs, trypan blue (Sigma) exclusion was used to determine viable cells. The expression of LMP2 protein in rAd-LMP2-DCs was analyzed by indirect immunofluorescence and FACS assays. In addition, DC phenotypes CD80, CD83, and CD86 were determined as well. Preparation of T lymphocytes. Sterile nylon-wool isolation column (Wako, Japan) was soaked in complete RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin for 1 h at 37 C. Then the nonadherent cells isolated from peripheral blood mononuclear cells described previously were applied on the column and cultured for additional 1 h. T lymphocytes were eluted from the column with 10 ml RPMI 1640/10%FCS. Purity of about 90% was obtained with this method. Flow cytometric analysis of cell populations. DCs were collected and resuspended in cold FACS buffer (phosphate-buffered saline with 0.2% BSA and 0.09% sodium azide). Cells were immunostained with fluorescein isothiocyanate (FITC) conjugated mouse anti-human CD80, CD83, and CD86 antibodies (eBioscience, USA). Corresponding FITC immunoglobulin G (IgG) isotype control antibody (eBioscience, USA) was used. A total of 1 · 106 cells were incubated overnight at 4 C with antibodies. The cells were then washed once with FACS buffer, resuspended, and phenotyped on a FACScan (Becton–Dickinson, USA). An intracellular staining method was used for the detection of LMP2 proteins in rAd-LMP2-DCs. Mature DCs were fixed in 2% paraformaldehyde. Cell membranes were permeated in 2% Triton X-100 (Amresco, USA) and then incubated with LMP2 rabbit multiclonal antibody (obtained from Institute for Viral Disease Control and Prevention of Chinese CDC) at 4 C overnight. After washing with PBS twice, the cells were immunostained with FITC-conjugated goat anti-rabbit IgG (Sigma) for 30 min at 37 C. The cells were then washed once with FACS buffer, resuspended, and analyzed on a FACScan. Lymphocyte proliferation assays. Lymphocyte proliferation assays were performed by using rAd-LMP2-DCs, untransfected DCs and CNE-2 cells as stimulator cells and T lymphocytes as responder cells. Stimulator cells were incubated with Mitomycin C (MMC) at 25 lg/ml at 37 C for 30 min and then washed with PBS twice. T lymphocytes isolated from the peripheral blood mononuclear cells were plated in 96-well flat-bottomed culture plate (Costar, USA) at 5 · 105 cells per well. Then stimulators were added and co-cultured with responders at ratios of 1:5, 1:10, 1:20, 1:50, 1:100, and 1:200 for 96 h at 37 C in 5% CO2. T cells incubated in medium alone served as control. The cells were then incubated with 5 mg/ml metrizamide (MTT; Sigma) 20 ll per well for 4 h. The supernatant was removed and 150 ll dimethyl sulfoxide (DMSO; Amresco, USA) was added to each well and agitated for 10 min to fully dissolve the crystals. Absorbance was measured at 570 nm on automatic ELISA reader (TRITURUS). All determinations were carried out in triplicate and repeated four times. Stimulation index (SI) was calculated as follows: SI = (experimental blank)/(control blank). Induction of CTLs by transfected DCs. T cells were harvested by nylonwool separation as described previously. T cells (1 · 106) were co-cultured with rAd-LMP2-DCs (5 · 104) in a 24-well tissue culture plate in 1 ml RPMI 1640/20% FCS at 37 C in 5% CO2. IL-2 was added at a final concentration of 40 IU/ml to all wells 3 days later and every 2–3 days thereafter. Responding T cells were re-stimulated weekly for 2 weeks with transfected DC at a responder T cell-to-stimulator DC ratio of 20:1. The CTLs were then collected and used as the effector cells in CTL assays against CNE-2 cells. Cytotoxicity assays. The target cells were placed in 96-well tissue culture plates at 1 · 104 cells per well and co-cultured with effector cells (CTLs) at the ratio of 1:5, 1:10, and 1:20 for 48 h at 37 C in 5% CO2. The cytotoxic activities were determined by MTT assay. Freshly prepared and filtered 20 ll metrizamide (5 mg/ml) was added to each well, and the cells were continuously cultured for 4 h. The supernatant was removed and 150 ll dimethyl sulfoxide was added to each well and agitated for 10 min to fully dissolve the crystals. Absorbance was measured at 570 nm on automatic ELISA reader (TRITURUS). All determinations were carried

Y. Pan et al. / Biochemical and Biophysical Research Communications 347 (2006) 551–557 out in triplicate and repeated four times. Experiments were performed in triplicate. The percentage of specific cytotoxicity was calculated as [(experimental minimal)/(maximal minimal)] · 100. Target cells incubated in medium alone or in medium containing 1% Triton X-100 were used to determine minimal and maximal cytotoxicity, respectively. T cells separated from the peripheral blood mononuclear cells by sterile nylonwool isolation column as described previously and the untransfected DCs were used as controls, respectively. Analysis of LMP2-specific CTLs populations. T cells were stimulated with rAd-LMP2-DCs weekly as described previously. After two rounds of stimulations, the induced LMP2-specific CTLs were collected on day 14 and then resuspended in cold FACS buffer. Cells were immunostained with FITC/PE/PE-cyanine5 (Cy5) conjugated mouse anti-human CD4/ CD8/CD3 antibodies (Jingmei Biotech, China). Corresponding mouse FITC/PE/PE-Cy5 IgG isotype control antibody (Jingmei Biotech, China) was used. A total of 1 · 106 cells were incubated with antibodies for 30 min at 37 C. The cells were then washed once with FACS buffer, resuspended, and bidimensional analyzed with a FACScan. Statistical analysis. SPSS11.0 was used for data variation analysis; p values less than 0.05 were considered statistically significant.

Results Morphological features of rAd-LMP2-DCs Fully morphologic differentiation of mature DCs activated by calcium ionophore required 72–96 h of culture. The rAd-LMP2-DCs retained typical morphological features of DCs (Fig. 1). Whilst untreated PBMCs maintained their rounded, smooth surface morphology and appeared as dispersed, nonadherent cells in culture, rAd-LMP2DCs predominantly gathered in clusters as nonadherent or loosely adherent cells with a larger cell surface and irregular shape. Phenotype of transfected DCs To determine whether mature DCs transfected with rAd-LMP2 expressed co-stimulatory molecules, mature DCs with or without rAd-LMP2 transfection were analyzed for co-stimulatory molecules (CD80 and CD86) and DC activation marker (CD83). We found these immunophenotypic alterations occurred promptly within the first 20–40 h of culture with calcium ionophore A23187. Adenoviruses transfection of mature DCs did

A

not result in significant increases or decreases in CD83, CD80, or CD86 expression. Data shown in Fig. 2 are representative of three independent experiments that produced similar results. Expression of LMP2 in transfected DCs Immature DCs were transfected with rAd-LMP2 at MOI 200 for 2 h. The transfected cells were cultured in the presence of calcium ionophore and rhGM-CSF for additional 48 h. When mature rAd-LMP2-DCs were analyzed by flow cytometry, the percentage of transfected DCs expressing LMP2 was 84.54%, which suggested efficient gene transfer (Fig. 3). Stimulation of T lymphocytes by rAd-LMP2- DCs It was found that anti-tumor T cells were generated by a single stimulation with mature DCs transfected with rAdLMP2. The rAd-LMP2- DCs were more potent stimulators of T lymphocytes than untransfected DCs (p < 0.05) or CNE-2 cells (p < 0.01), respectively. The effect was enhanced with higher ratio of rAd-LMP2-DCs to T cells (Fig. 4). Cytotoxicity assays After stimulating twice with rAd-LMP2-DCs, highly LMP2-specific anti-tumor CTLs could be induced. The cytotoxic activity was enhanced with increased ratio of effector-to-target cells. MTT assay showed that cytotoxic activity in rAd-LMP2-DCs group was higher than that in untransfected DCs (p < 0.05) and T cell groups (p < 0.01), respectively (Fig. 5). Effect of stimulation on cytotoxicity T cells were stimulated by rAd-LMP2-DCs weekly. The induced CTLs were harvested as effector cells. These effector cells were used against target cells in cytotoxicity assay as previously described on day 7, 14, and 21. The results showed that LMP2-specific cytotoxicity elicited by only a single stimulation of transfected DCs was higher than those by T

B

PBMCs

553

C

untransfected DCs

rAd-LMP2-DCs

Fig. 1. Morphological characters of PBMCs (A), untransfected DCs (B), and transfected DCs (C). After 96 h culture, PBMCs were grown by suspension. DCs with or without transfection appeared similar morphology with long dendritic projection. Photomicrographs were taken with inverted phase contrast microscope under 200· magnifications.

554

Y. Pan et al. / Biochemical and Biophysical Research Communications 347 (2006) 551–557

Mature untransfected DCs

Mature transfected DCs

Fig. 2. Phenotype of mature DCs with or without rAd-LMP2 transfection. Cells were incubated with FITC, conjugated mAbs against CD80, CD86, and CD83. The result showed that mature untransfected DCs (top panel) with expressions of CD80, CD83, and CD86 were 86.32, 85.73, and 86.27%, respectively; mature transfected DCs (bottom panel) were 81.54, 87.48, and 88.37%, respectively. 35

rAd-LMP2-DC untransfected DC CNE-2

Stimulation Index(SI)

30

Fig. 3. Expression of LMP2 in untreated and transfected DCs. Flow cytometry indicated expression of LMP2 in rAd-LMP2-DCs (B) was 84.54 and 1.57% in untreated DCs (A).

cell group (p < 0.01) and untransfected DC group (p < 0.05). Further more, the cytotoxicity could augment with repeated stimulations. Compared with that on day 7, the specific cytotoxicity was evidently higher on day 14 in all groups, respectively (p < 0.01), but there was no significant difference between those on day14 and on day 21 (p > 0.05) in untransfected DC group and rAd-LMP2-DC group (Fig. 6). The experiments indicated that two rounds of stimulation were enough to induce potent specific cytotoxicity. Flow cytometric analysis of LMP2-specific CTLs populations LMP2-specific CTLs induced by rAd-LMP2-DCs on day 14 were collected and analyzed with flow

25 20 15 10 5 0 1:5

1:10

1:20

1:50

1:100

1:200

Ratio of stimulator cell to T cell

Fig. 4. T lymphocytes proliferation reaction stimulated by rAd-LMP2DCs, untransfected DCs, and CNE-2. Mature DCs transfected with rAdLMP2 for 48 h were collected and co-cultured with T cells for 96 h. Specific CTLs were detected by MTT assay. The results are expressed as means ± SD of three replicates. Data indicate that rAd-LMP2-DCs were potent stimulators of lymphocyte than untransfected DCs (p < 0.05) or CNE-2 (p < 0.01), respectively.

cytometry. We found that the CTLs consisted of CD4+ and CD8+ T cells simultaneously. The component of CD8+ T cell was slightly larger than that of CD4+ T cell (Fig. 7).

Y. Pan et al. / Biochemical and Biophysical Research Communications 347 (2006) 551–557

55 50

E G F

*# *#

45

Cytotoxity(%)

40 35

*#

30 25 20 15 10 5 0 5:1

10:1

20:1

Ratio of effector to target cell

Fig. 5. Cytotoxicity of CTLs against nasopharyngeal carcinoma cells. The CNE-2 cells were placed in 96-well tissue culture plates at 1 · 104 per well and co-cultured with effector cells at the ratio of 1:5, 1:10, and 1:20 for 48 h. Percentage cytotoxicity (mean ± SD of three replicates) was determined by MTT assay. *p < 0.01 vs. T cell group; #p < 0.05 vs. untransfected DC group.

Discussion Recently, malignancy-associated viruses are used as potential targets for immunotherapeutic vaccines aiming to stimulate T-cell responses against viral antigens expressed in tumor cells [12–14]. Here we have shown that the induction of primary antigen-specific CTL responses in vitro by human PBMCs-derived DCs adenovirally transfected with LMP2 gene, a subdominant antigen in EBV-associated NPC cells. DCs are professional APCs that play a critical role in the activation of the immune response to antigen. Mature DCs express high levels of co-stimulatory mole-

cules, necessary components of T-cell activation by APCs, which must occur in conjunction with MHC-restricted presentation of the antigen to the T-cell receptor. The co-stimulatory molecules identified on DCs with their respective T-cell receptors are CD54, CD80, CD83, CD86, CD40, and CD40 ligand. In the present study, the co-stimulatory molecules CD80, CD86, and CD83 did not change significantly after rAd-LMP2 modification of DCs compared with untreated DCs, which demonstrated adenovirus transfection had little effect on DC maturation and antigen-presenting function. Recent laboratory observations indicated that pharmacologic agents that mobilize intracellular calcium can be used to enhance APC functions in human PBMCs [15,16]. The phospholipase C (PLC)-calcium signaling pathway is involved in the maturation of DCs induced by the agonists such as calcium ionophore A23187. A23187 can pump extracellular calcium into the cell. The increased cytoplasmic calcium concentration induced by A23187 could cause calcium-induced calcium release from intracellular stores. On the other hand, increased cytoplasmic calcium levels may induce positive feedback that activates PLC, which causes the second messengers inositol 1,4,5-triphosphate (IP3) liberating from the plasma membrane. Thus, calcium release from IP3-gated stores induces the maturation of DCs. In our study, we tried to use calcium ionophore as the main agent to generate DCs more effectively from human PBMCs and examined the characteristics of DCs including cellular morphology and APC function. Our results showed that fully morphologic differentiation of DCs activated by calcium ionophore required 72–96 h of culture, and immunophenotypic alterations occurred promptly within the first 20–40 h of culture after CI treatment, including upregulation of CD80 and CD86 expression, and de novo expression of the DC-associated Day21

60

T cell untransfected DC rAd-LMP2-DC

Day14

55

555

50 45

Cytotoxity(%)

40

Day7

35 30 25 20 15 10 5 0 5

10

20

5

10

20

5

10

20

Ratio of effector to target cell

Fig. 6. Effect of different rounds of stimulation on cytotoxicity. T cells were stimulated by rAd-LMP2-DCs weekly, and the induced CTLs were harvested as effector cells. These effector cells were used against target cells in cytotoxicity assay on day 7, 14, and 21. Cytotoxicity on day 14 was obviously higher than that on day 7 (p < 0.01), but there was no significant difference between those on day 14 and on day 21 (p > 0.05).

556

Y. Pan et al. / Biochemical and Biophysical Research Communications 347 (2006) 551–557

Fig. 7. Flow cytometric analysis of LMP2-specific CTLs populations. The percentage of CD3+, CD4+, and CD8+ T cells was 61.73, 36.47, and 46.18%, respectively.

activation marker CD83. Such rapid activation kinetics contrasted to the much slower activation (needed about 9–10 days) observed when PBMCs were treated with cytokine combinations such as rhGM-CSF, rhIL-4, and rhTNF-a [5,6]. Gene transfer is an attractive means to affect the immunostimulatory properties of DCs. We have used a gene-based vaccination strategy by using DCs expressing the tumor antigen to elicit a potent therapeutic anti-tumor immunity. This approach has apparent advantages over protein- or peptide-based immunization [17]. Tumor associated antigen (TAA) gene expression in DCs causes endogenous processing and presentation of multiple and/or undefined antigenic peptides independent of MHC alleles. Furthermore, specific T cell-mediated immunity may be stimulated by vaccine-involved APC without prior knowledge of responder MHC haplotypes or of relevant MHC class I- or class II-restricted peptide epitopes. Although a variety of vectors are available for gene transfer to DCs, recombinant adenovirus is most efficient. Adenovirus vector is a highly efficient and reproducible method of gene transfer. Indeed, several studies have shown that successful adenoviral gene transfer into human DCs resulted in induction of a T-cell response against tumor [18,19]. Our results showed that the expression of LMP2 in transfected DCs reached a high level of 84.54%, which indicated efficient gene transfection. Currently, published reports showed that malignancyassociated virus antigen could induce specific antitumor CTL. EBV is a herpesvirus commonly associated with malignancies such as Hodgkin disease (HD), T-cell lymphoma, and NPC, particularly in immunocompromised hosts. EBV elicits a strong cytotoxic T lymphocyte (CTL) response directed against a broad range of viral antigens that are involved in the control and regulation of latency and in the induction of proliferation and transformation [20]. EBV-associated NPC, a high-inci-

dence tumor in southern China, expresses a limited set of EBV proteins. Only latent class II EBV antigens such as EBNA1 and LMP1, LMP2 can be detected on NPC cells. Among these three antigens, LMP1 is an NPC-associated viral oncogene [21], and EBNA1 is an abundant source of HLA class II-restricted CD4+ T-cell epitopes that contains a Gly-Ala repeat sequence, which can interrupt the presentation of it through HLA class I-restricted subway to T cells [22,23]. LMP2 is a source of subdominant CD8+ T-cell epitopes presented by HLA class I alleles common in the Chinese population [24,25]. In some studies, EBV transformed B lymphoblastoid cell lines (LCLs) have been used to induce EBV-specific CTLs. Adoptive transfer of EBV-specific CTLs has been successfully applied in the treatment of EBV associated post-transplant lymphoproliferative disease [26,27]. Nevertheless, application of this approach to EBV-associated NPC is difficult, because LCLs focus T cell expansion on immunodominant EBV antigens such as the latent EBNA3A, 3B, and 3C that are not expressed in EBV-associated NPC. On the other hand, in adoptive immunotherapy LCLs elicited only EBVspecific memory T cell responses but not native T-cell responses [28]. In our study, we demonstrated that DCs transfected with LMP2 by adenovirus vector were able to stimulate enhanced T-cell proliferation and LMP2-specific cytotoxic T-cell responses in vitro. Analyzing the populations of CTLs elicited by rAd-LMP2DCs, we found LMP2-specific CTLs consisted of both CD4+ and CD8+ T cells simultaneously. Our result was similar to the reports of CTLs induced by DCs transfected with LMP2a in experiment of HD treatment [26]. The specific CTLs lysed carcinoma cells maybe by both MHC class I- and MHC class II-restricted mechanisms. In summary, the results demonstrate that vaccination using DCs simultaneously transfected with malignancy-associated virus antigens can elicit potent CTL response and provide a potential immunotherapy strategy for EBV-associated NPC.

Y. Pan et al. / Biochemical and Biophysical Research Communications 347 (2006) 551–557

Acknowledgments This work was supported by the National ‘‘863’’ Project of China (NO. 2003AA216071) and the National Natural Science Foundation of China (NO. 30270520).

[14]

Appendix A. Supplementary data

[15]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc. 2006.05.214.

[16]

References [1] G. Niedobitek, Epstein-Barr virus infection in the pathogenesis of nasopharyngeal carcinoma, J. Clin. Pathol. 53 (2000) 248–254. [2] R.M. Steinman, The dendritic cell system and its role in immunogenicity, Ann. Rev. Immunol. 9 (1991) 271–296. [3] J. Banchereau, R. Steinman, Dendritic cells and the control of immunity, Nature (Lond.) 392 (1998) 245–252. [4] T. Tomohide, G. Andrea, D.R. Paul, J.S. Walter, Interleukin 18 gene transfer expands the repertoire of antitumor Th1-type immunity elicited by dendritic cell-based vaccines in association with enhanced therapeutic efficacy, Cancer Res. 62 (2002) 5853–5858. [5] L. Jenne, J.F. Arrighi, H. Jonuleit, et al., Dendritic cells containing apoptotic melanoma cells prime human CD8+ T cells for efficient tumor cell lysis, Cancer Res. 60 (2000) 4446–4452. [6] M. Nouri-Shirazi, J. Banchereau, D. Bell, et al., Dendritic cells capture killed tumor cells and present their antigens to elicit tumorspecific immune responses, J. Immunol. 165 (2000) 3797–3803. [7] K.C. Bagley, S.F. Abdelwahab, R.G. Tuskan, G.K. Lewis, Calcium signaling through phospholipase C activates dendritic cells to mature and is necessary for the activation and maturation of dendritic cells induced by diverse agonists, Clin. Diagn. Lab. Immunol. 11 (2004) 77–82. [8] E. Sadovnikova, E.N. Parovichnikova, E.L. Semikina, et al., Adhesion capacity and integrin expression by dendritic-like cells generated from acute myeloid leukemia blasts by calcium ionophore treatment, Exp. Hematol. 32 (2004) 563–570. [9] H.J. Du, L. Zhou, J.M. Zuo, et al., A study on killing effect of cytotoxic T cell activated by LMP2 peptides in nasopharyngeal carcinoma cells, J. Oncol. 10 (2004) 92–94. [10] M.A. Rosenfeld, K. Yoshimura, B.C. Trapnell, et al., In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium, Cell 68 (1992) 143–155. [11] N. Mittereder, K.L. March, B.C. Trapnell, Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy, J. Virol. 70 (1996) 7498–7509. [12] S. Gottschalk, O.L. Edwards, U. Sili, et al., Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies, Blood 101 (2003) 1905–1912. [13] H.J. Wagner, U. Sili, B. Gahn, et al., Expansion of EBV latent membrane protein 2a specific cytotoxic T cells for the adoptive

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

557

immunotherapy of EBV latency type 2 malignancies: influence of recombinant IL12 and IL15, Cytotherapy 5 (2003) 231–240. L. Santodonato, G. D’Agostino, R. Nisini, et al., Monocyte-derived dendritic cells generated after a short-term culture with IFN-alpha and granulocyte-macrophage colony-stimulating factor stimulate a potent Epstein-Barr virus-specific CD8+ T cell response, J. Immunol. 170 (2003) 5195–5202. T.M. Westers, A.G. Stam, R.J. Scheper, et al., Rapid generation of antigen-presenting cells from leukaemic blasts in acute myeloid leukaemia, Cancer Immunol. Immunother. 52 (2003) 17–27. M. Waclavicek, A. Berer, L. Oehler, et al., Calcium ionophore: a single reagent for the differentiation of primary human acute myelogenous leukaemia cells towards dendritic cells, Br. J. Haematol. 114 (2001) 466–473. S.S. Diebold, M. Cotten, N. Koch, M. Zenke, MHC class II presentation of endogenously expressed antigens by transfected dendritic cells, Gene Ther. 8 (2001) 487–493. L.F. Paul, L. Christie, M.A. Jennifer, et al., Efficacy of CD40 ligand gene therapy in malignant mesothelioma, Am. J. Resp. Cell Mol. Biol. 29 (2003) 321–330. M.L. Jay, M. Ali, Y. Reiko, et al., Adenovirus vector-mediated overexpression of a truncated form of the p65 nuclear factor jB cDNA in dendritic cells enhances their function resulting in immunemediated suppression of preexisting murine tumors, Clin. Cancer Res. 8 (2002) 3561–3569. R. Khanna, S.R. Burrows, Role of cytotoxic T lymphocytes in Epstein-Barr virus-associated diseases, Annu. Rev. Microbiol. 54 (2000) 19–48. J.M. Burrows, L. Bromham, M. Woolfit, et al., Selection pressuredriven evolution of the Epstein-Barr virus-encoded oncogene LMP1 in virus isolates from Southeast Asia, J. Virol. 78 (2004) 7131–7137. C. Munz, K.L. Bickham, M. Subklewe, Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1, J. Exp. Med. 191 (2000) 1649–1660. R. Khanna, J. Tellam, J. Duraiswamy, et al., Immunotherapeutic strategies for EBV-associated malignancies, Trends Mol. Med. 7 (2001) 270–276. C.L. Lin, W.F. Lo, T.H. Lee, et al., Immunization with Epstein-Barr Virus (EBV) peptide-pulsed dendritic cells induces functional CD8+ T-cell immunity and may lead to tumor regression in patients with EBV-positive nasopharyngeal carcinoma, Cancer Res. 62 (2002) 6952–6958. G.S. Taylor, T.A. Haigh, N.H. Gudgeon, et al., Dual stimulation of Epstein-Barr Virus (EBV)-specific CD4+- and CD8+-T-cell responses by a chimeric antigen construct: potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma, J. Virol. 78 (2004) 768–778. C.M. Bollard, K.C. Straathof, M.H. Huls, et al., The generation and characterization of LMP2-specific CTLs for use as adoptive transfer from patients with relapsed EBV-positive Hodgkin disease, J. Immunother. 27 (2004) 317–327. D. Chua, J. Huang, B. Zheng, et al., Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma, Int. J. Cancer. 94 (2001) 73–80. W. Herr, E. Ranieri, W. Olson, et al., Mature dendritic cells pulsed with freeze-thaw cell lysates define an effective in vitro vaccine designed to elicit EBV-specific CD4(+) and CD8(+) T lymphocyte responses, Blood 96 (2000) 1857–1864.