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Human Monocyte-Derived Dendritic Cells Pulsed with

Wild-Type p53 Protein Efficiently Induce CTLs against

p53 Overexpressing Human Cancer Cells

Naoyuki Tokunaga,1 Takayoshi Murakami,1

Yoshikatsu Endo,1 Masahiko Nishizaki,1

Shunsuke Kagawa,2 Noriaki Tanaka,1

and Toshiyoshi Fujiwara1,2

1Division of Surgical Oncology, Department of Surgery, OkayamaUniversity Graduate School of Medicine and Dentistry, and2Center for Gene and Cell Therapy, Okayama University Hospital,Okayama, Japan

ABSTRACT

Purpose: Dendritic cells are the most potent antigen-

presenting cells for initiating cellular immune responses.

Dendritic cells are attractive immunoregulatory cells for cancer

immunotherapy, and their efficacy has been investigated in

clinical trials. The tumor suppressor gene p53 is pivotal in the

regulation of apoptosis, and p53-based immunization is an

attractive approach to cancer immunotherapy because of the

accumulation of p53 protein in malignant but not in normal

cells. It has been shown that dendritic cells transduced with

an adenoviral wild-type p53 (wt-p53) construct mediate the

antitumor immune responses against p53-overexpressing

tumor cells. We examined whether monocyte-derived human

dendritic cells pulsed with the purified full-length wt-p53

protein were also capable of inducing the specific antitumor

responses against p53-overexpressing tumors in vitro.

Experimental Design: Immature dendritic cells generated

in the presence of interleukin-4 and granulocyte/macrophage

colony-stimulating factor from monocytes of HLA-A2- or

HLA-A24-positive healthy individuals were pulsed with the

purified p53 protein. Uptake of p53 protein by human

dendritic cells was assessed by Western blotting and immuno-

histochemical staining using anti-p53 antibody. Induction of

p53-specific CTL response was also evaluated by the cytotoxic

assay against p53-overexpressing human tumor cells.

Results: Both Western blot and immunohistochemical

analysis showed the accumulation of p53 protein in human

immature dendritic cells. T cells obtained from HLA-A2- or

HLA-A24-positive healthy donors were stimulated twice with

p53 protein-pulsed dendritic cells and then applied to the

cytotoxicity assay against p53-overexpressing target cells.

The CTL activity was specific for p53-overexpressing tumor

cells and MHC class I restricted. Moreover, the CTL activity

generated by p53 protein-pulsed dendritic cells was nearly

identical with that induced by adenoviral wt-p53-transduced

dendritic cells.

Conclusions: Our results indicate that monocyte-

derived human dendritic cells pulsed with the wt-p53 protein

could induce the specific antitumor effect against p53-

overexpressing tumors and that this in vitro model offers a

new and more simple approach to the development of p53-

based immunotherapy.

INTRODUCTION

Dendritic cells are highly effective antigen-presenting cells

and play a central role in the induction of primary immune

responses against tumor-associated antigens (1). The exceptional

ability of dendritic cells to stimulate T cells is attributed to their

ability to take up and present antigens, to secrete cytokines, and

to express high levels of immunostimulatory molecules, such as

B7.1 (CD80), B7.2 (CD86), and intercellular adhesion molecule-

1 (CD54). Critical to the antigen-presenting function of dendritic

cells is the fact that they can present tumor-associated antigens in

the context of both MHC class I and II; thus, they can stimulate

both CTL and helper T cells (2–4). Because of the advantageous

character of dendritic cells compared with other antigen-

presenting cells, numerous studies for vaccines based on

dendritic cells have examined their efficacy in animal models

(5, 6) as well as in human clinical trials (7–9).

Much attention has been directed to the problem of how and

what antigens should be pulsed to the dendritic cells. In this

regard, p53 protein is one of the most attractive candidates for

dendritic cell–based immunotherapy, because this protein is

found abundantly in f50% of human malignancies but not in

normal tissues (10). The p53 tumor suppressor gene plays a key

role in cell growth control and differentiation (11). This gene

containing missense mutations is often associated with a

prolonged half-life, resulting in accumulation of the inactivated

protein within the nuclei and cytoplasm of cancer cells. It has been

shown that overexpression of the mutant p53 protein results in the

presentation of p53 peptides by MHC class I molecules on the

surface of tumor cells (12, 13). This may lead to the generation of

multiple epitopes that could be recognized by CTLs. Indeed,

previous studies have reported the successful generation of

antitumor immune responses against tumors bearing a mutant p53

protein following either vaccination in vivo or stimulation of

peripheral blood mononuclear cells (PBMC) in vitro using mutant

or wild-type p53 (wt-p53) peptide-pulsed dendritic cells (14–16).

However, this approach to cancer immunotherapy has some

serious limitations. One strategy to overcome these limitations

is to use the entire p53 sequences as an antigen.

Received 5/4/04; revised 9/2/04; accepted 9/13/04.Grant support: Ministry of Education, Science, and Culture, Japan andMinistry of Health and Welfare, Japan.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Requests for reprints: Toshiyoshi Fujiwara, Center for Gene and CellTherapy, Okayama University Hospital, 2-5-1 Shikata-cho, Okayama700-8558, Japan. Phone: 81-86-235-7997; Fax: 81-86-235-7884; E-mail:[email protected].

#2005 American Association for Cancer Research.

Vol. 11, 1312–1318, February 1, 2005 Clinical Cancer Research1312

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One of the best vehicles for delivery of the entire p53

sequences to dendritic cells is the use of virus vectors encoding

full-length wt-p53 gene. In fact, recent studies have reported that

dendritic cells transduced with a recombinant replication-

deficient adenovirus vector expressing wt-p53 gene were able

to effectively present the recombinant protein antigen (p53) and

could induce specific immune response against p53 protein

in vitro and in vivo (17, 18). However, in terms of the clinical

use, this effective approach with virus vectors still leaves several

critical problems concerning safety. Accordingly, in this study,

we showed that monocyte-derived human dendritic cells pulsed

with purified full-length wt-p53 protein could also generate p53-

specific CTLs in vitro . In addition, the CTL activity generated

by whole p53-protein pulsed dendritic cells was almost the same

as that by adenoviral wt-p53 (Ad-p53)– transduced dendritic

cells. These results offer a new effective approach to the p53-

based immunotherapy and provide justification for continuing

the preclinical and clinical development of full-length wt-p53-

pulsed dendritic cells.

MATERIALS AND METHODS

Tumor Cell Lines and Reagents. Tumor cell lines

SW620 and KATO-III were maintained in RPMI 1640

supplemented with 10% FCS, 100 units/mL penicillin, and

100 mg/mL streptomycin, referred to as complete medium.

Both cell lines are HLA-A2/A24 positive. The transformed

embryonic kidney cell line 293 was grown in DMEM with

high glucose concentration (4.5 g/L), supplemented with 10%

FCS, 100 units/mL penicillin, and 100 mg/mL streptomycin.

The 293 cells were used for the production of adenovirus

vectors. Recombinant human cytokines granulocyte/macro-

phage colony-stimulating factor, interleukin-4 (IL-4), tumor

necrosis factor-a (TNF-a), and IL-7 were purchased from

Genzyme Techne, (Minneapolis, MN) and IL-2 was from

Roche (Mannheim, Germany). 51Cr sodium chromate was

obtained from NEN Life Science Products (Boston, MA).

p53 Protein and Adenovirus Vectors. Full-length wt-p53

protein as well as mutant p53 protein (His273 mutation) were

kindly provided by Kyowa Hakko (Tokyo, Japan). The

recombinant, replication-deficient adenovirus vector, Ad-p53,

encodes full-length human wt-p53 cDNA. This virus was

obtained by lysis of infected 293 cells. Titers of viral stocks

were determined with a plaque-forming assay using 293 cells.

Cell Isolation. Monocyte-derived dendritic cells and T

cells were obtained from peripheral blood of HLA-A2- or HLA-

A24-positive healthy volunteers. In brief, mononuclear cells were

isolated by sedimentation over Ficoll-Hypaque and were

subsequently allowed to adhere in culture flasks for 1 hour at

37jC. Nonadherent cells were separated from the rest of

mononuclear cells by gentle washing and were cryopreserved in

RPMI 1640 with 20% FCS and 10% DMSO for further use. The

remaining (adherent) cells were cultured for 6 days in the presence

of granulocyte/macrophage colony-stimulating factor (50 ng/mL)

or IL-4 (50 ng/mL) and used as immature dendritic cells.

Generation of Anti-p53 CTLs. p53-specific CTLs were

generated as follows. On day 6, immature dendritic cells

were incubated with wt-p53 protein at a final concentration of

0.2 Ag/mL at 37jC for 24 hours. Subsequently, the protein-

pulsed dendritic cells were activated with TNF-a (50 ng/mL)

for 72 hours. In another experiments, immature dendritic cells

were infected with Ad-p53 at the multiplicity of infection of 100

plaque-forming units per cell for 24 hours. Nonadherent

mononuclear cells were then cocultured with p53 protein-pulsed

or Ad-p53-transfected dendritic cells at a ratio of 10:1 in the wells

of a 24-well plate in a final volume of 2.0 mL/well complete

medium containing IL-2 (10 units/mL) and IL-7 (5 ng/mL).

After 7 days of culture, the responder cells were collected

and restimulated with p53 protein-pulsed or Ad-p53-transfected

dendritic cells again. Fifty percent of the medium were replaced

on days 3 and 5 after each stimulation by complete medium

supplemented with IL-2 (10 units/mL) and IL-7 (5 ng/mL). On

14 day of coculture, the responder cells were harvested and

assessed for cytolytic activity in a standard 6-hour chromium

release assay.

Cytotoxicity Assay. Standard 6-hour chromium release

assay was done to measure cytolysis. Target cells were

incubated with 100 mCi Na251CrO4 for 1 hour, washed, and

added to the wells of U-bottomed 96-well plates (5 � 103 cells

per well). Effector T cells were then added in triplicates to give

various E:T ratios ranging from 80:1 to 10:1. After a 6-hour

incubation, the supernatants were collected, and the radioac-

tivity was measured in a scintillation counter. The percentage

of specific lysis was calculated according to the formula: %

Specific lysis = [(experimental cpm � spontaneous cpm) /

(maximal cpm � spontaneous cpm) � 100].

Flow Cytometric Analysis. The cell surface expression

of dendritic cell markers was assessed by flow cytometric

analysis. Antibodies used to evaluate the phenotype of dendritic

cell were anti-HLA-DR-FITC, anti-CD80-FITC, anti-CD86-PE,

and anti-CD83-PE (ImmunoTech, Marseilles, France). Dendritic

cells were stained for 20 minutes at 4jC and analyzed by the

FACScan using Cell Quest software. Ten thousand cells were

examined for each determination. Autologous T cells cultured

with p53 protein-pulsed dendritic cells were also analyzed for the

phenotype by flow cytometric analysis using anti-CD4 and anti-

CD8 antibodies that were purchased from Becton Dickinson

(San Jose, CA).

Immunostaining. Cytospin preparations of p53 protein-

pulsed dendritic cells and tumor cell lines were fixed in 4%

paraformaldehyde and stained with the primary antibody

against human p53 (Ab-2, Oncogene Science, Manhasset, NY)

overnight at 4jC. This staining was done according to the

protocol provided by the manufacturer of Envision/HRP System

(DAKO, Carpinteria, CA).

Measurement of Cytokine. Supernatants of the culture

were recovered and assayed in duplicate by ELISA using

commercially available reagents (PharMingen, San Diego, CA).

The concentration of cytokine in the supernatant was determined

by regression analysis.

Statistical Analysis. Student’s t test was used to compare

differences in CTL reactivities against different tumor cell lines.

Statistical significance was defined when P < 0.05.

RESULTS

Preparation of Dendritic Cells and p53 Protein.

Representative phenotypic characteristics of immature and

mature dendritic cells that were used in these studies are shown

in Fig. 1A . Flow cytometric analysis showed that additional

Clinical Cancer Research 1313



maturation of immature dendritic cells in TNF-a (25 ng/mL) and

prostaglandin E2 (1 Ag/mL) led to the expression of HLA-DR,

CD80, CD86, and CD83 markers on the cell surface. We next

did Western blot analysis using antibody against human p53 to

confirm the stability of full-length wt-p53 protein. Both full-

length wild-type and mutated p53 proteins could be equally

detected at the same level of expression, suggesting that wt-p53

protein is quite stable in vitro (data not shown).

Uptake of Full-length p53 Protein by Human Dendritic

Cells. To confirm that human immature dendritic cell ingests

the full-length wt-p53 protein spontaneously, we carried out

immunohistologic analysis. Immunohistochemistry showed that

immature dendritic cells pulsed with wt-p53 protein at a final

concentration of 0.2 Ag/mL for 24 hours exhibited intense

cytoplasmic and nuclear staining of p53 protein (Fig. 2A). We

also coincubated wt-p53 protein with immature dendritic cells

for various periods at various concentrations to determine the

optimal condition. The uptake of p53 protein was quantitatively

equivalent in immature dendritic cells pulsed with 0.2 Ag/mL of

p53 for 24 hours and immature dendritic cells pulsed with 1.0

Ag/mL of p53 for 3 hours (Fig. 2B). However, the viability of

dendritic cells pulsed with p53 at 1.0 Ag/mL for 24 hours was

reduced (data not shown). These results suggest that immature

dendritic cells can effectively ingest the full-length wt-p53

protein when pulsed at 0.2 Ag/mL for 24 hours.

Influence of wt-p53 Protein on Differentiation and

Activation of Human Monocyte-Derived Dendritic Cells.

To evaluate the effect of wt-p53 protein on the differentiation

and activation of human immature dendritic cells, we analyzed

the cell surface phenotype of dendritic cells pulsed with wt-p53

protein at 0.2 Ag/mL for 24 hours or control immature dendritic

cells by flow cytometry. The intensity of expression of surface

markers, such as costimulatory and adhesion molecules,

remained stable on wt-p53 protein-pulsed dendritic cells

compared with that on control immature dendritic cells (Fig. 3).

We next examined whether exposure to TNF-a (50 ng/mL)

could induce maturation on dendritic cells pulsed with wt-p53

protein. TNF-a increased in the expression levels of surface

markers, including HLA-DR, CD80, CD86, and CD83, in a

time-dependent manner (Fig. 3). These results suggest that the

wt-p53 protein had no apparent influence on dendritic cell

maturation.

We also examined whether p53 protein could alter the

cytokine production profiles of dendritic cells. After a culture

period of 3 days, the supernatants of control immature dendritic

cells and wt-p53 protein-pulsed immature dendritic cells were

harvested, and the levels of IFN-g in the supernatants were

measured by ELISA. Both control and p53 protein-pulsed

immature dendritic cells produced low to undetectable amounts

of IFN-g (0.101 F 0.048 and 0.303 F 0.247 pg/mL,

Fig. 1 Exposure of dendritic cells (DC) to TNF-a and prostaglandin E2 modifies the expression of cell surface markers. On day 5 of culture in thepresence of granulocyte/macrophage colony-stimulating factor plus IL-4, dendritic cells were harvested at immature state. Maturation of dendritic cellswas induced by the addition of TNF-a (25 ng/mL) and prostaglandin E2 (1 Ag/mL). After 72-hour incubation, changes in expression of cell surfacemarkers (HLA-DR, CD80, CD86, and CD83) were analyzed by flow cytometry. Left, isotype-matched negative controls. Mature dendritic cells up-regulated all four surface markers. Percentages of positive cells.

Fig. 2 Immature dendritic cells uptake pulsed wt-p53 protein. A, after6-day culture of adherent PBMCs with granulocyte/macrophage colony-stimulating factor and IL-4, immature dendritic cells were pulsed withwt-p53 protein at a final concentration of 0.2 Ag/mL for 24 hours. Cellswere then stained with anti-p53 antibody. Note the intense cytoplasmicand nuclear staining in p53 protein-pulsed immature dendritic cells. Left,untreated dendritic cells; right, p53 protein-pulsed dendritic cells.Original magnification, � 200. B, optimal condition for uptake of p53protein by immature dendritic cells. Dendritic cells were pulsed with wt-p53 protein at either 0.2 Ag/mL for 24 hours (left) or 1.0 Ag/mL for 3hours (right) and then immunostained for p53. No apparent differencewas noted. Original magnification, � 400.

Immunotherapy with p53-Overexpressing Dendritic Cells1314



respectively), indicating that p53 protein had no apparent effect

on cytokine production of dendritic cells.

Induction of p53-Specific CTL Response against Tumor

Cells. We selected two HLA-A2/A24 human tumor cell lines,

SW620 and KATO-III, as targets for p53-specific CTLs. Human

colorectal cancer cell line SW620 exhibits overexpression of p53

caused by the specific mutation at codon 273, whereas human

gastric cancer cell line KATO-III is p53 null. Immunohisto-

chemistry confirmed that SW620 cells are p53 positive and

KATO-III cells are p53 negative (Fig. 4). SW620 and KATO-III

cells were positive for MHC class I expression (19, 20).

p53-specific CTLs were generated with p53 protein-pulsed

dendritic cells from autologous T cells of three healthy HLA-A2+

donors, and the CTL response against two target cell lines was

assessed by a standard 6-hour 51Cr release assay. As shown in

Fig. 5A , effector T cells stimulated with wt-p53 protein-pulsed

dendritic cells could effectively lyse p53-overexpressing SW620

cells. On the other hand, p53-null KATO-III cells were only

minimally lysed. Although mononuclear cells primed by Ad-

p53-transduced dendritic cells also specifically lysed p53-

overexpressing SW620 cells, the lytic activity was almost the

same as that stimulated with wt-p53 protein-pulsed dendritic

cells. Phenotypic analysis of effector T cells indicated that the

percentages of CD4+ and CD8+ cells were 68.13% and 24.41%,

respectively. To further verify the specificity of the CTL

response, the cytotoxicities of p53-specific CTLs, human

lymphokine-activated killer (LAK) cells, and untreated T cells

were examined against SW620 and KATO-III cell lines. LAK

cells were generated from nonadherent mononuclear cells in the

presence of IL-2 (100 units/mL) for 3 days. The lytic activity of

CTLs induced by p53 protein-pulsed dendritic cells against

SW620 cells was comparable with that of LAK cells. In contrast,

LAK cells effectively killed KATO-III cells, whereas cells were

only minimally lysed by CTLs (Fig. 5B). These results were

consistent with CTLs primed by Ad-p53-infected dendritic cells.

To show the broad applicability of p53 protein-pulsed

dendritic cells, we examined whether p53-specific CTLs could

be generated from HLA-A24+ healthy volunteers. Besides the

HLA-A2 allele, HLA-A24 is one of the most frequently expressed

HLA-A alleles. CTL responses against mutant p53-expressing

SW620 cells, but not against p53-null KATO-III cells, were

induced with wt-p53-pulsed dendritic cells obtained from two

HLA-A24+ healthy volunteers (Fig. 6). These results suggest that

wt-p53 protein-pulsed dendritic cells are likely to present

multiple p53 epitopes on different class I HLA molecules, such

as HLA-A24 and HLA-A2.

DISCUSSION

The p53 mutations or functional inactivation in human

cancers lead to accumulation of p53 protein, whereas normal cells

have very low levels of p53 expression. This makes p53 protein a

potent target for immunotherapy of human cancer. Here, we show

Fig. 3 Effect of p53 proteinon dendritic cell phenotypes.Immature dendri t ic cel lsobtained from monocytes werepulsed with or without wt-p53protein at 0.2 Ag/mL for 24hours, incubated for another 48hours, and then analyzed forexpression of the surfacemarkers by flow cytometry.p53 protein had no apparenteffect on the expression ofcostimulatory molecules. Den-dritic cells pulsed with wt-p53protein were stimulated withTNF-a (50 ng/mL) for 48 or72 hours, and the dendritic cellphenotypes were assessed byflow cytometry. Left, isotype-matched negative controls. Af-ter 72-hour culture, the expres-sion of cell surface markers wasfully changed. Percentages ofpositive cells.

Fig. 4 Immunohistochemical detection of p53 protein overexpressionwith a specific antibody against human p53 in SW620 human colorectalcancer cell line and KATO-III human gastric cancer cell line. Nuclearexpression of overexpressing p53 protein was detected in SW620 cellsbut not in KATO-III cells.




that CTLs induced by in vitro activation with dendritic cells

pulsed with full-length wt-p53 protein elicited effective killing of

p53-overexpressing human tumor cell lines. Several groups have

reported previously that MHC class I–bound p53 peptides

induced p53-specific CTLs, and several clinical trials are

currently in progress (14–16); these CTLs are however mostly

much less effective against p53-overexpressing tumor cells in

spite of very effective cell lysis against peptide-pulsed targets.

These observations suggest the limitation of peptide-based

vaccine therapy and led us to examine whether monocyte-derived

dendritic cells pulsed with the entire recombinant sequence of p53

protein efficiently induce p53-specific CTLs in vitro .

Recently, Nikitina et al. (17, 18) have reported that dendritic

cells transduced with full-length wt-p53 gene using an adenovirus

vector could generate a specific antitumor immune response.

Overexpression of the entire sequence of wt-p53 in dendritic cells

might present several different epitopes not only on the MHC

class I but also on the MHC class II that induces CD4+ T-cell

immune response. This strategy may be quite attractive; the

application of adenovirus vector however made its clinical use

difficult concerning safety. In addition, immunomodulatory

proteins coded by an adenoviral vector might be expressed

in dendritic cells, recognized as antigens compounded into cells,

and scarcely presented on the MHC class II molecules. Thus, we

suggest an alternative method of p53 protein-based immuno-

therapy using dendritic cells pulsed with full-length wt-p53

protein. After incubation with whole p53 protein, dendritic cells

may process the endocytosed p53 proteins into peptides that are

bound to MHC class II molecules and be presented on the cell

surface for stimulation of CD4+ T helper lymphocytes. In

addition to this classic mechanism of antigen presentation,

dendritic cells turned out to be capable of processing exogenous

proteins by an alternative pathway leading to peptide presentation

on MHC class I molecules, which is called cross-presentation.

Because immature dendritic cells are highly effective in

taking up and processing antigens compared with mature

dendritic cells, we used monocyte-derived immature dendritic

cells to be pulsed with wt-p53 protein. Immunohistochemical

analysis showed a sufficient ingestion of pulsed p53 protein

(Fig. 2). Generation of antitumor immunity by dendritic cells is

intimately linked to dendritic cell maturation stage. Recent

reports have shown that recombinant adenovirus infection

induces partial maturation of dendritic cells via a nuclear

factor-nB–dependent pathway (21). We also found that infection

of adenovirus slightly up-regulated the expression of costimu-

latory and MHC class II surface molecule 3 days after infection

(data not shown). On the other hand, p53 protein-pulsed

dendritic cells showed no distinct alteration of the expression

of surface markers (Fig. 3). Accordingly, we concluded that the

maturation of p53 protein-pulsed dendritic cells would be

indispensable. We next asked which activators would be suitable

for maturation of p53 protein-pulsed dendritic cells. Many

dendritic cell activators, including CD40 ligand, various

cytokines, bacterial or viral products, calcium ionophores, and

others (22–27), had been already reported to up-regulate the

costimulatory molecule expression, secrete IL-12, and effectively

present antigens. Among these dendritic cell activators, TNF-a

is one of the most commonly used cytokines for this purpose.

Although TNF-a alone is not sufficiently potent to cause full

maturation of dendritic cells, it was selected as a dendritic cell

stimulator in this study based on clinical applicability.

Fig. 5 Cytolytic reactivity of PBMC-derived CTLs against SW620 andKATO-III human cancer cells. Mean F SD from three wells. A, cell lysisactivity of T cells from three HLA-A2+ healthy volunteers stimulatedwith Ad-p53-transfected dendritic cells (left) or wt-p53 protein-pulseddendritic cells (right) against target cells was assessed by 6-hour standard51Cr release assay. Each experiment was done in triplicates. Points, meanat four different E:T ratios; bars, SD. B, CTLs were compared with LAKcells and untreated T cells, which served as positive and negativecontrols, respectively. Top, Ad-p53-transfected dendritic cells; bottom,wt-p53 protein-pulsed dendritic cells.

Fig. 6 Cytolytic reactivity of T cells obtained from two HLA-A24+

healthy donors exposed to wt-p53 protein-pulsed dendritic cells was alsoassessed by CTL assay. Points, mean from three wells; bars, SD.




Another important area of our investigation was to define

the appropriate maturational state of dendritic cells. It has been

reported that Ad-p53-transduced dendritic cells activated with

CD40 ligand were able to break tolerance to self-p53 protein

and induce potent antitumor response in mice, whereas

nonactivated p53-expressing dendritic cells were unable to

overcome the tolerance (17). Moreover, recent studies have

shown that dendritic cells that ingest apoptotic tumor cells or

pulsed with tumor cell lysate could also effectively generate

tumor-specific T-cell response in vitro when mature (28, 29). In

our study, flow cytometric analysis showed that treatment with

TNF-a induced maturation of p53 protein-pulsed dendritic cells

in a time-dependent manner (Fig. 3). Accordingly, we used p53-

expressing dendritic cells stimulated with TNF-a for 3 days in

subsequent experiments. However, it has been reported recently

that dendritic cells exhibited a direct cytotoxic effect on tumor

cells and that immature dendritic cells undergoing maturation

induced a much stronger tumor-specific growth inhibitory effect

than immature dendritic cells or mature dendritic cells through

TNF-a-dependent and TNF-a-independent mechanisms (30).

We showed that intratumoral injection of nonactivated bone

marrow–derived dendritic cells transduced with adenovirus

expressing murine wt-p53 gene could mediate a greater systemic

immune response against tumor cells than those of dendritic cell

alone or control vector-transduced dendritic cells (31). Immature

dendritic cells delivered locally at the site of tumors can be

expected to ingest adjacent tumor cells and present additional

tumor antigens in vivo , which may be facilitated by maturing

dendritic cell–mediated direct cell lysis and tumor-specific CTL

responses. Therefore, dendritic cells undergoing maturation

might be more appropriate than fully mature dendritic cells,

when a clinical trial of intratumoral administration of p53

protein-pulsed dendritic cells is tested.

To confirm the successful generation of CTLs from PBMCof

a healthy HLA-A2-positive donor in vitro with p53 protein-pulsed

dendritic cells that recognize the wt-p53 epitopes, we examined

the lytic activity against target the human tumor cell lines SW620

and KATO-III. These HLA-A2- and HLA-A24-positive tumors

showed either accumulation of mutant p53 molecules with a point

mutation at codon 273 or expression of a p53-null genotype with

no p53 accumulation (Fig. 4). We detected high CTL responses

against mutant p53-expressing SW620 cells after 7-day in vitro

stimulation of PBMC with p53 protein-pulsed dendritic cells;

however, p53-null KATO-III cells were not effectively killed by

CTLs (Fig. 5A). The observation that LAK cells lysed KATO-III

target cells (Fig. 5B) suggest that the low killing activity of CTLs

against KATO-III cells is due to the lack of p53 antigen

presentation. In other words, the effector cells are likely to be

p53-specific CTLs. We also showed that p53-specific CTLs could

be generated from PBMC of a HLA-A24-positive donor in vitro

by using p53 protein-pulsed dendritic cells in the same in vitro

stimulationmethod (Fig. 6). Based on these findings, we speculate

that multiple p53 peptides are presented on HLA class I molecules

and recognized by autologous CTLs. Our findings indicate that

dendritic cells pulsed with p53 protein can overcome the

limitations of peptide-based vaccines, such as a priori knowledge

of the patient’s HLA haplotype to select appropriate peptides

compatible with that particular haplotype. Therefore, it may

permit a more potent immune response involving the presentation

of epitopes from all possible restriction elements. To answer this

question, we are currently investigating the lytic activity of p53-

specific CTLs against target tumor cells pulsed with different p53-

derived peptides.

Taken together, our study indicate that monocyte-derived

human dendritic cells pulsed with purified full-length wt-p53

protein can generate p53-specific CTLs whose lytic activity

against p53-overexpressing target cells is similar to that induced

by stimulation with Ad-p53-transduced dendritic cells in vitro .

To the best of our knowledge, this is the first report describing

the CTL induction with full-length wt-p53 protein-pulsed

dendritic cells. Our data offer a new and potentially valuable

option of p53-based immunotherapy for human cancer.

ACKNOWLEDGMENTSWe thank Drs. Marimo Sato, Takuya Tsunoda, and Hideaki Tahara

(University of Tokyo, Tokyo, Japan) for the helpful discussion and

Yoshiko Shirakiya for technical support.

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2005;11:1312-1318. Clin Cancer Res Naoyuki Tokunaga, Takayoshi Murakami, Yoshikatsu Endo, et al. Overexpressing Human Cancer CellsWild-Type p53 Protein Efficiently Induce CTLs against p53 Human Monocyte-Derived Dendritic Cells Pulsed with

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