High immunogenic potential of p53 mRNA-transfected dendritic cells in patients with primary breast cancer.

Abstract

As pre-existent immunity might be a reflection of an emerging anticancer response, the demonstration of spontaneous T-cell responses against tumor associated antigens (TAAs) in cancer patients may be beneficial before clinical development of dendritic cell (DC)-based cancer vaccines, because it will help to identify likely responders to TAAs among patients who qualify and may benefit from this form of immune therapy. This study aimed to determine pre-existent T-cell reactivity against the tumor suppressor protein p53 in breast cancer patients (BCP) at the time point of primary diagnosis. After a short-term stimulation with autologous wt p53 mRNA-transfected DCs, IFN-γ enzyme-linked immunosorbent spot (ELISPOT) analysis revealed p53-reactive T cells in the peripheral blood of more than 40% (15 of 36) of the tested patients. Both CD4(+) and CD8(+) p53-specific T cells secreted IFN-γ after stimulation with p53-transfected DCs. Interestingly, more than 72% (13 of 18) of patients with high p53 (p53(high)) expression in tumors were able to mount a p53-specific IFN-γ T-cell response, in contrast to only 10% (1 of 10) of healthy donors and 11% (2 of 18) of patients with low or absent p53 (p53(low)) expression in tumors. Furthermore, significantly higher secretion of IL-2 was detected in peripheral blood mononuclear cells after stimulation with p53-transfected DCs from patients with p53(high) tumor expression compared to patients with p53(low) tumor expression, whereas secretion of IL-10 was predominant in the latter group. The high frequency of spontaneous wt p53-reactive T cells detected in the peripheral blood of primary BCP with accumulation of p53 in tumor provides a rationale to consider DCs transfected with mRNA encoding wt p53 for clinical investigation in these patients.

Full text

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High immunogenic potential of p53 mRNA-transfected dendritic cells in
patients with primary breast cancer


Özcan Met1, 2*, Eva Balslev3, Henrik Flyger4, Inge Marie Svane1, 2;

1Center for Cancer Immune Therapy (CCIT), Department of Hematology, 2Department of
Oncology, 3Department of Pathology and 4Department of Breast Surgery, University
Hospital Herlev, Copenhagen, Denmark






Running title: T-cell response in breast cancer against p53-transfected DCs



*Correspondence:
Özcan Met
Center for Cancer Immune Therapy
Department of Hematology, 65Q9
University Hospital Herlev
Herlev Ringvej 75
Copenhagen, 2730 Herlev
Denmark

E-Mail: ozcmet01@heh.regionh.dk
Phone: +45 44884000 - 89229
Fax: +45 44884153
peer-00555001, version 1 - 12 Jan 2011
Author manuscript, published in "Breast Cancer Research and Treatment 125, 2 (2010) 395-406"
DOI : 10.1007/s10549-010-0844-9

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Abstract

As pre-existent immunity might be a reflection of an emerging anticancer response, the
demonstration of spontaneous T-cell responses against tumor associated antigens (TAAs)
in cancer patients may be beneficial before clinical development of DC-based cancer
vaccines, because it will help identify likely responders to TAAs among patients who
qualify and may benefit from this form of immune therapy. This study aimed to
determine pre-existent T-cell reactivity against the tumor suppressor protein p53 in breast
cancer patients at the time point of primary diagnosis. After a short-term stimulation with
autologous wt p53 mRNA-transfected DCs, IFN-γ ELISPOT analysis revealed p53-
reactive T cells in the peripheral blood of more than 40% (15 of 36) of the tested patients.
Both CD4+ and CD8+ p53-specific T cells secreted IFN-γ after stimulation with p53-
transfected DCs. Interestingly, more than 72 % (13 of 18) of patients with high p53
(p53high) expression in tumors were able to mount a p53-specific IFN-γ T-cell response,
in contrast to only 10 % (1 of 10) of healthy donors and 11 % (2 of 18) of patients with
low or absent p53 (p53low) expression in tumors. Furthermore, significantly higher
secretion of IL-2 was detected in PBMCs after stimulation with p53-transfected DCs
from patients with p53high tumor expression compared to patients with p53low tumor
expression, whereas secretion of IL-10 was predominant in the latter group. The high
frequency of spontaneous wt p53-reactive T cells detected in the peripheral blood of
primary breast cancer patients with accumulation of p53 in tumor provides a rationale to
consider DCs transfected with mRNA encoding wt p53 for clinical investigation in these
patients.

Key words: dendritic cells; mRNA transfection; immunotherapy; breast cancer; tumor
associated antigens

Abbreviations used: DCs, dendritic cells; TAAs, tumor associated antigens; PBMCs,
peripheral blood mononuclear cells; MFI, mean fluorescence intensity; ELISPOT,
enzyme-linked immunosorbent spot; BCP, breast cancer patients; HD, healthy donors;
Abs, antibodies

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Introduction

The re-emerging view of cancer immunosurveillance is based on findings that malignant
tumors can be naturally recognized by the host’s immune system and induce spontaneous
tumor associated antigen (TAA)-specific T-cell responses [1, 2]. Over the last decade,
DCs have been subject to intense investigation as cellular adjuvant in cancer vaccines,
based on the discovery that vaccination with DCs charged with TAAs is a potent strategy
to elicit protective immunity in tumor-bearings hosts [3]. The rationale behind DC-based
vaccination approaches is to stimulate effective cytotoxic T-cell responses in cancer
patients by isolating DCs from the patient, load them with TAAs and inject the antigen-
loaded cells back into the patient. This has been accomplished by incubating DCs with
peptides, proteins or by transfecting the cells with DNA constructs. Another appealing
approach is to engineer DCs to synthesize tumor epitopes endogenously by transfecting
with mRNA encoding TAAs [4]. The use of electroporation to release mRNA as
translatable genetic material provides excellent means of TAA transfer [5].
Information on the incidence and magnitude of TAA-specific immune responses is
important for identifying which TAAs are naturally targeted by the immune system and
for understanding why natural immunity fails to eradicate tumors. TAAs that either play a
role in the oncogenic process or promote cancer cell survival are favorable
immunotherapeutic targets [6]. Among the several dozen of such antigens, the tumor
suppressor protein p53 is particularly attractive as it is essential for the maintenance of
the non-tumorigenic phenotype of cells [7]. In addition, the resultant over-expression of
p53, triggered by the prolonged half-life of p53 protein as a result of mutation in the p53
gene, provides multiple potentially immunogenic p53 epitopes, which can be exploited as
a target in, for example, the immunotherapy of breast cancer [8]. Although studies have
shown that breast cancer is an immunogenic tumor, information concerning the extent
and prevalence of pre-existent TAA-specific T-cell responses in breast cancer patients
remains contradictory [9-11]. This may be explained by the predominant focus on MHC-I
and single peptide epitopes responses. Furthermore, it is very likely that tumor-specific
immune responses, like responses against infectious diseases, consist of multiple
effectors including CD4+ T cells, CD8+ T cells, and antibodies. Since transfection of DCs
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with TAA-encoding mRNA provides the possibility of presenting a broad spectrum of
TAA epitopes and thus allows the determination of polyclonal T-cell responses against
the transfected TAA, we therefore, tested for the presence and frequencies of p53-specific
T cells in breast cancer patients at the time point of primary diagnosis. Our data suggest
that wt p53 mRNA-transfected DCs could be a useful immunotherapeutic strategy
particularly in breast cancer patients with p53 expressing tumors.





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Materials and Methods

Patients and blood samples

Blood samples were obtained after informed consent from patients with primary breast
cancer before surgery. Patients enrolled in this study had not received any neoadjuvant or
adjuvant chemotherapy, hormone therapy, or radiotherapy before sample collection.
Peripheral blood mononuclear cells (PBMCs) from breast cancer patients were isolated
from heparinized blood by Lymphoprep (Nycomed, Oslo, Norway) density
centrifugation, washed twice with PBS and once with X-VIVO 15 (Lonza, Verviers,
Belgium) prior to cryopreservation in human AB-serum (PAA, Pasching, Austria) and
10% DMSO. Aliquots of 1x107 cells/ml were stored in -140°C and were used as
stimulators or responder cells in experiments. Viability of thawed cells was above 80 %.
PBMCs from healthy donors were separated and cryopreserved as previously described
[12].

Generation of monocyte-derived DCs

DCs were generated by plating thawed PBMCs in Nunclon dishes (Nunc, Biotech Line,
Slangerup, Denmark) at 5x106 cells/well in culture medium consisting of X-VIVO 15
(Lonza) supplemented with 1% glutamax and 5% heat-inactivated human AB-serum
(PAA). Cells were allowed to adhere for 1.5 h in 5% CO2 at 37°C, and nonadherent cells
were removed. Adherent cells were washed and suspended in culture medium
supplemented with 250 IU/ml rh-IL-4 (CellGenix, Freiburg, Germany) and 1000 IU/ml
GM-CSF (CellGenix), and incubated for six days for differentiation of DCs. On day 6, a
cocktail consisting of 1000 U/ml IL-1β (CellGenix), 1000 U/ml TNF-α (CellGenix), 1000
U/ml IL-6 and 1 μg/ml PGE2 (Minprostin, Pharmacia/Pfizer, Ballerup, Denmark) was
added to the cell culture and DCs were allowed to mature for 24 h. Mature DCs [13] were
electroporated the following day and used for experimental analysis as specified in the
text. Alternatively, immature DCs were electroporated with mRNA day 7, transferred
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back to the culture with added maturation cocktail and used for experimental analysis as
indicated.

Production of in vitro transcribed mRNA

The wt p53 cDNA from pCMV-p53 (Clontech-Takara Bio Europe, Paris, France) was
cloned into pSP73-SphA64 (kindly provided by Professor E Gilboa, Duke University
Medical Center, Durham, NC) using HindIII and EcoRI restriction sites. The pSP73-
Sph/p53/A64 plasmid was propagated in Escherichia coli competent cells (Invitrogen,
Paisley, UK) and was purified using CompactPrep Plasmid Maxi Kit (Qiagen, Hilden,
Germany). Prior to serving as DNA templates for in vitro transcription, pSP73-
Sph/p53/A64 was linearized with SpeI and purified using Wizard DNA Clean-Up
System (Promega). The in vitro transcription was performed with mMESSAGE
mMACHINE T7 Ultra kit (Ambion, Austin TX, USA) and mRNA was purified with
MEGAclear kit (Ambion) according to the manufacturer’s instructions. The
mRNA length, concentration and purity were evaluated with the Agilent 2100
Bioanalyzer (Agilent Technologies, Palo Alto CA, USA), using RNA 6000 Nano
LabChip Kit (Agilent Technologies) according to the manufacturer’s instructions. Data
analysis was performed with 2100 Bioanalyzer software (Agilent Technologies).

Electroporation of DCs

DCs were washed twice, suspended in Opti-MEM medium (Invitrogen) and adjusted to a
final cell density of 5x106 cells/ml. The cell suspension (250 μl) was preincubated in a 4-
mm gap electroporation cuvette for five min on ice. Ten μg of mRNA encoding p53 was
transferred to the cuvette and DCs were pulsed using a BTX 830 square-wave
electroporator (Harvard Apparatus, Holliston MA, USA). Electroporation settings were
adjusted to a single pulse, 500 V, 2 ms. After electroporation, DCs were rested in 37°C
and subsequently transferred to prewarmed culture medium supplemented with GM-CSF
and IL-4 with or without maturation cocktail as described above. Transfected DCs [12]
were further incubated in humidified atmosphere with 5% CO2 and used for experimental
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analysis as specified in the text. All mock-transfected DCs used as controls underwent
electroporation following the same conditions as described above.

Flow cytometric analysis

Messenger RNA-transfected DCs generated from healthy donors were checked for p53-
expression by intracellular FACS. After electroporation, DCs were permeabilized with
Cytofix/Cytoperm solution (BD Biosciences, San Jose CA, USA) and stained with
antibodies according to manufacturer’s instructions. FITC conjugated mouse-anti-human
p53 (Abcam, Cambridge, UK) antibody were used for detection of p53 protein in mRNA-
transfected DCs. Mock-transfected DCs were also stained and used as negative control.
Fluorescence analysis was performed with a FACSCalibur flow cytometer using Cell
Quest software (BD BioSciences). The expression level (MFI) was determined as the
ratio of the relative MFI of DCs electroporated with p53 mRNA divided by the relative
MFI of mock-electroporated DCs.

Immunohistochemical staining for p53

Sections of formalin-fixed, paraffin-embedded tissue from the primary breast carcinoma
were cut and stained with antibody against p53 DO7, (DAKO, Glostrup, Denmark), 1:50.
In brief, the slides were immersed in citrate buffer solution for antigen retrieval and
boiled in microwave for 10 min and washed in buffer solution (TBS). They were
incubated with primary antibody for 1 h at room temperature and then washed in TBS.
After 1 h of incubation in the secondary antibody, the sections were incubated with
streptavidin-biotin-complex (DAKO). A multi-block including several different tissues
was used for positive and negative controls. For estimation of positive reaction, only the
strongly stained nuclei were counted as a percentage of all tumor nuclei. Less than 5%
were estimated as low p53 expression or as negative [14].

Stimulation of p53-specific T cells
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DCs were transfected with p53 mRNA and used as stimulators in order to determine the
presence and frequencies of p53-specific T cells in breast cancer patients. To extend the
sensitivity of the ELISPOT assay, PBMCs were stimulated once in vitro before analysis
[15]. Transfected DCs were cocultured with autologous PBMCs (nonadherent fraction
after adherence step for DC generation) at a DC:PBMC ratio of 1:10. The following day,
40 units/ml IL-2 were added to the cultures and cells were tested for p53-reactivity in the
ELISPOT assay on day 7. In other experiments (as specified in the text) CD4+ and CD8+
T lymphocytes were purified from PBMCs by CD4+ and CD8+ T cell isolation kit
(Miltenyi Biotec, Bergisch Gladbach, Germany) prior to stimulation with p53-
transfected DCs and IFN-γ ELISPOT analysis as described above.

ELISPOT assay

The ELISPOT assay was used to quantify p53-specific IFN-γ-releasing effector T cells.
Briefly, nitrocellulosebottomed 96-well plates (MultiScreen MAIP N4550, Millipore,
Billerica MA, USA) were coated with anti-IFN-γ Ab (1-D1K; Mabtech, Nacka Strand,
Sweden). The wells were washed, blocked with X-VIVO 15 medium and the effector T
cells were added in duplicates at different cell concentrations. DCs transfected with p53
mRNA were added to the wells at a DC/T-cell ratio of 1:10. The plates were incubated
overnight at 37°C/5% CO2. The following day, medium was discarded and the wells were
washed prior to addition of biotinylated secondary Ab (7-B6-1-Biotin, Mabtech). The
plates were incubated at room temperature (RT) for 2 h, washed, and Streptavidin-ALP
(Mabtech) was added to each well. Plates were incubated at RT for 1 h and the enzyme
substrate BCIP/NBTplus (Mabtech) was added to each well and incubated at RT for 5-
10 min. Upon appearance of dark purple spots, the reaction was terminated by washing
with tap water. Spots were counted using ImmunoSpot 2.0 Analyzer (CTL, Cleveland
OH, USA) and the frequency of p53-specific T cells were calculated from the number of
spot-forming cells.

Multiplexed ELISA (Luminex) assays
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PBMCs from eight patients with high p53 (p53high) tumor expression and eight patients
with low or absent p53 (p53low) tumor expression were stimulated with p53-transfected
DCs and supernatants from overnight ELISPOT cultures were collected and analyzed
using the human Th1/Th2 5-plex panel kit (Invitrogen), which measured IFN-γ, IL-2, IL-
4, IL-5 and IL-10, and Luminex 200 (Luminex Corporation, Austin TX, USA)
according to the manufacturer’s instructions. PBMCs from eight healthy donors
underwent same procedure as described above. Data analysis was performed with the
STarStation v2.3 software (Applied Cytometry Systems, Sheffield, UK)

Analysis of anti-p53 antibodies in serum

p53-specific IgG titers were quantified using the commercially available p53 ELISAPLUS
(Autoantibody) Kit (Calbiochem/Merck Chemicals Ltd., Nottingham, UK) according to
manufacturer’s instructions and evaluated using at least four serial dilutions.
Results for each serum sample were calculated by determining the relative p53 index and
internal controls provided by the manufacturers were used to establish a “cutoff” level
(0.15 U of p53 binding activity/μl patient serum). Samples were always assayed in
duplicate. Absorbance was determined on a spectrophotometer at a wavelength of 450
nm against a reference filter of 620 nm in order to compensate for differences in the
material of the microtiter plate. Samples were designated positive for p53 antibody when
the absorbance were > 20 % cutoff value, whereas samples were designated negative for
p53 antibody when the absorbance were < cutoff value.

Statistics

Significant differences between sample means in ELISPOT and Luminex assays were
determined with Mann-Whitney’s test and results were considered significant
when p < 0.05. Statistical analysis of the p53-specific IgG response in relation to IL-10
expression was carried out by a Fisher’s exact test and results were considered significant
when p < 0.05.
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Results

Expression kinetic of p53 in DCs electroporated before or after maturation

Before electroporation, the quality of the in vitro-transcribed wt p53 mRNA was
analyzed with the Agilent 2100 Bioanalyzer. This system provides a more precise
description of the integrity and quantity of the mRNA sample compared to the
traditionally used agarose gel electrophoresis and spectrophotometry. Fig. 1a depicts the
result of a representative electrophoretic chip run. The electropherogram and gel-like
image provide data on size and concentration of mRNA, and also show the presence of
degradation products in the mRNA sample. Only highly pure mRNA preparations were
used for electroporation of DCs.
We then checked the expression profile of p53 protein by intracellular staining and flow
cytometry in mature electroporated DCs or immature DCs electroporated prior to
maturation. In DCs electroporated after maturation, the intracellular expression of p53
was substantial as early as 2 h after transfection, followed by an increased expression
level which reached a maximum at 6 h post-transfection (Fig. 1b). The mean fluorescence
intensity remained 3-4 folds higher in mature electroporated DCs at early time points (2-6
h) compared to DCs electroporated prior to maturation. At the time point of maximum
intracellular expression, more than 95 % of transfected DCs were stained positive for p53
expression (Fig. 1a, histogram plot). However, while the expression of p53 declined
rapidly reaching an almost undetectable level 10 h after electroporation of mature DCs, a
more sustained expression of p53 was observed in DCs electroporated before maturation,
showing intermediate level expression of p53 12 h post-transfection. The relatively high
turn-over of p53 in transfected DCs is in marked contrast to the expression of enhanced
green fluorescence protein (EGFP), often used as a model for TAAs in DC-transfection
studies, which is stable 5 days after transfection [12].

T-cell reactivity against p53 mRNA-transfected DCs in patients with
primary breast cancer
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The presence of T cells specific for p53 in peripheral blood of primary breast cancer
patients was determined at the time point of first diagnosis. For this purpose, PBMCs
from both breast cancer patients and healthy donors were stimulated with autologous
mature DCs transfected with p53 mRNA and p53-specific T cells were subsequently
analyzed by ELISPOT IFN-γ release assay. Before analysis, PBMCs were restimulated
once in vitro to extend the sensitivity of the assay.
Using this approach we were able to detect IFN-γ T-cell reactivity against p53-
transfected DC targets in 15 of 36 breast cancer patients (Fig. 2a and b). In contrast, only
one of 10 normal donors responded in this way to DCs transfected with p53 mRNA.
These data suggest a high frequency of pre-existing cellular response in breast cancer
patients against p53 at the time point of primary diagnosis.
To address which T-cell subpopulation was responsible for the secretion of IFN-γ, the T-
cell populations from seven responder patients were each separated into CD4+ and CD8+
T-cell fractions by magnetic bead technology prior to stimulation with p53-transfected
DCs and IFN-γ ELISPOT analysis. The purity of CD4+ and CD8+ T cells was routinely
found to be >90% as assessed by flow cytometry (data not shown). ELISPOT analysis
depicted in Fig. 2c demonstrates that both subtypes of p53-specific T cells secrete IFN-γ
after stimulation with DCs transfected with p53 mRNA.

T-cell reactivity against p53 is associated with the accumulation of p53 in
tumors from breast cancer patients

Data from our previous phase II study showed a higher frequency of p53-expression in
primary tumor samples from breast cancer patients attaining stable disease compared to
patients with progressive disease during p53-targeted DC-vaccination therapy [16].
Accordingly, we questioned whether the accumulation of p53 in the tumor from breast
cancer patients might influence the presence of IFN-γ T-cell reactivity against p53
mRNA-transfected DCs. For this purpose, immunohistochemical staining was done to
study the expression profile of p53 in surgically resected primary tumor specimens.
Immunohistochemical analysis of two representative tumor specimens is shown in Fig.
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3a. We selected 18 patients with p53high tumor expression in tumors and 18 patients with
p53low tumor expression, as measured by nuclear staining intensity (see Materials and
methods) for evaluation of IFN-γ T-cell reactivity against p53 mRNA-transfected DC
targets. As depicted in Fig. 3c, 13 out of 18 patients with p53high expression in tumors had
IFN-γ T-cell reactivity against p53-transfected DCs in contrast to only two of 18 patients
with p53low nuclear staining.
Thus, these data indicate that ex vivo IFN-γ T-cell response to p53-transfected DCs is
predominant in patients with over-expression of p53 in tumors.

Cytokine profile of T cells in breast cancer patients with high or low/absent
p53 expression in tumor

We then analyzed whether T-cell reactivity against p53-transfected DCs was associated
with a specific pattern of cytokine secretion. To this end, PBMCs from eight patients with
p53high tumor expression and eight patients with p53low tumor expression were stimulated
with p53-transfected DCs as described above. Supernatant were collected and analyzed
with Multiplex Luminex cytokine assays. The assay demonstrated that p53-transfected
DCs generally induced a significantly higher expression of the Th1-associated cytokine
IL-2 (Fig 4a and Table 1) in PBMCs from patients with p53high expression in tumors
compared to patients with p53low expression. In contrast, the secretion of IL-10 was more
frequent in the group of patients with low or absent p53 expression (Fig. 4b). No
significant difference of IL-4 and IL-5 secretion was detected among the patient groups
(data not shown).
We also tested these patients for the presence of p53-specific IgG antibodies in serum.
Four patients with p53high and only one patient with p53low tumor expression displayed
p53-specific IgG serum antibodies (Table 1). Interestingly, the presence of p53-specific
IgG antibodies correlated with in vitro secretion of IL-10 in patients with p53high tumors
(Table 1; p = 0.03, Fisher’s exact test).



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Discussion

The analysis of natural immune responses against TAAs in cancer patients is informative
as it can help identify antigens to which tolerance induction is non-existent, incomplete or
reversible. The prevalence of natural TAA-reactive T cells may also indicate that the
immune system of the patient is responsive to the tumor and could potentially be
exploited for tumor rejection [17]. Thus, such TAAs could be targeted in DC-based
treatment strategies because it may be easier to expand a memory pool of T
cells compared to generating new immunity. Data from a few selected patients suggest a
favorable clinical course in patients with pre-existent TAA-directed immunity [18, 19]. In
this regard, pre-existing T-cell responses to p53 have been reported in cancer patients
[20-22]. However, results have been conflicting with regard to the prevalence of pre-
existing TAA-directed T-cell responses in breast cancer [9-11, 23]. This might be due in
part to a predominant focus on single peptide epitope responses. The use of overlapping
peptide pools rather than single epitopes represents a more rational approach to analyzing
naturally occurring TAA-specific T cells in cancer patients. We have recently conducted
a phase II trial in which HLA-A2+ patients with progressive metastatic breast cancer were
vaccinated with autologous DCs pulsed with a cocktail of 3 wild-type and 3 modified
p53-peptides [16]. Yet, as mRNA-transfected DCs can induce a potent and broad (i.e.
polyclonal) response, we used this method to assess the prevalence of pre-existing T-cell
response against wt p53 in breast cancer patients at the time point of first diagnosis. After
a short-term stimulation with mRNA-transfected mature DCs, IFN-γ ELISPOT analyses
revealed p53-reactive T cells in the peripheral blood of more than 40% of the tested
patients. The p53 T cell response was particular prevailing in patients with p53high
expressing tumors. Several studies have shown higher frequency of TAA-specific T cells
in cancer patients with tumor expressing antigens compared to patients with no TAA
expression or healthy donors [24]. Our results demonstrate that even in early stage breast
cancer patients with small operable tumors accumulation of p53 in the tumor results in
the presence of relatively high frequencies of wt p53 epitope-specific T cells in the
circulation. The underlying premise here is that the accumulation of p53 protein due to
p53 mutation provides an opportunity for effective presentation of immunogenic wt p53
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epitopes to T cells and generation of wt p53-specific T cells in tumor-bearing hosts [7,
25]. While this may not always be the case, as indicated by results of Vierboom et al. and
Hofmann et al. [20, 26]; in the present study, IFN-γ responses to p53 mRNA-transfected
DCs were detected in PBMCs of more than 72 % of breast cancer patients with p53high
expression in tumors. In contrast, only 10 % of healthy donors and 11 % of patients with
p53low expression in tumors possessed this p53-specific reactivity. Because p53 is a
ubiquitous expressed protein [7] it is conceivable that a certain threshold level of
intracellular p53 protein must be exceeded in order to generate a p53-specific IFN-γ
response. However, although our data suggest that the expression levels of p53 are
clearly above this threshold in patients with over-expression of p53 in tumors, no direct
correlation to the distinct expression level was detected. The results support data from our
previous phase II study which showed a higher frequency of p53 expression in the
primary tumor samples from breast cancer patients attaining stable disease during p53
targeted DC therapy [16].
Unfortunately, because of sparse sample material, we were not able to address the
phenotype of the p53-specific T cells. However, the low frequency of p53-specific T-cell
response in healthy donors suggests that an antigen experienced phenotype is elicited by
tumor-driven immune activation [27, 28]. Although our data demonstrate that breast
cancer patients can elicit strong immune responses, it is also apparent that these responses
are not necessarily protective against tumor progression. What determines the variation
among cancer patients in terms of efficacy and intensity of antitumor immunity remains
to be elucidated and could be related to mechanisms such as the action of regulatory T
cells [29], Fas-mediated apoptosis induction in CTLs [30] and antigen down-regulation
by the tumor [31]. Further investigation is required to more accurately define the level of
p53 protein expression in tumors necessary for T-cell recognition and activation. Doing
so, will help to identify likely responders to p53 epitopes on transfected DCs and thereby
to select patients that may benefit from this form of immune therapy.
In a previous work [32], Nikitina et al. transduced DCs with p53 gene using an
adenovirus vector and were able to stimulate anti-p53 T cell response in eight of nine
patients with advanced stages of lung or head and neck cancer. The authors also
demonstrated highly specific T-cell responses against tumors with accumulation of p53.
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Still, even though this strategy appears to be efficient, it may also have negative impact
on DC functions including the risk of integration of viral gene into the host genome. In
addition, immunodominant viral products could suppress an immune response against the
transgene, which also restricts the application of multi-vaccinations [33].
Our data also show that upon activation with p53-transfected DCs, the secretion of the
Th1-associated cytokine IL-2 was considerably more prevalent in patients with p53high
expression in tumors compared to patients with p53low expression. In contrast,
significantly higher secretion of the Th2-associated cytokine IL-10 was detected in
patients with p53low tumor expression. The latter finding was unexpected and could be
explained by lack of sufficient amounts of p53 for cross presentation which in turn may
fail to induce immediate effector functions mediated by CTLs resulting in the
predominance of the Th2 arm of the p53-specific immune response (Spiotto MT et al.
Immunity 2002). There was also a significant correlation between p53-specific IgG
serum antibodies in patients which had an IL-10 response when stimulated with p53
mRNA-transfected DCs but, surprisingly this correlation was only found in patients with
p53high tumor expression, whereas, only one of the seven IL-10 responsive p53low patients
possessed p53 antibodies. Clearly, more extensive studies need to be conducted in order
to clarify any potential disease-associated Th1/Th2 bias in response to p53-transfected
DCs in patients with p53high compared to p53low tumor expression.
It has previously been shown that antibodies against p53 are associated with a poor
prognosis [34]. Reconciling with this, immunotherapeutic approaches that are able to
shift the balance towards Th1 polarization may be essential for more effective
immunotherapy in patients with advanced disease [35, 36].

Several studies clearly indicates that an optimal antitumor immune response will require
the concomitant activation of both the CD4+ and CD8+ T-cell arm of the immune
response [37, 38]. Thus, a potential drawback of mRNA transfection of DCs could be that
the endogenously expressed antigens derived from the transfected mRNA are
preferentially channeled into the MHC-I processing pathway and activate CD8+ but not
CD4+ T cells. As a result, vaccination with mRNA-transfected DCs would be deficient in
the stimulation of the CD4+ T-cell arm of the immune response [39]. However, recently it
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