Download Superiority of the ear pinna over muscle tissue as site for

Gene Therapy (1998) 5, 789–797
 1998 Stockton Press All rights reserved 0969-7128/98 $12.00
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Superiority of the ear pinna over muscle tissue as site
for DNA vaccination
¨
P Forg1,2, P von Hoegen1,3, W Dalemans3 and V Schirrmacher1
1
German Cancer Research Center (DKFZ), Division of Cellular Immunology, Im Neuenheimer Feld 280, D-69120 Heidelberg,
Germany
Three different vaccination sites were compared for
efficiency of immunization with naked DNA. Using the bacterial lacZ gene as a model, all three sites of the mouse
(skeletal muscle, dermis of abdominal skin or of the ear
pinna) could express the gene product ␤-gal but varied in
expression time with muscle tissue showing the longest
expression. Expression time, however, did not correlate
with immune response intensity. The ear pinna was by far
the most effective and muscle the least effective priming
site for specific humoral and cytotoxic T cell-mediated
immune responses. Following intra-pinna DNA inoculation,
␤-gal expressing cells were detectable around the injection
site and in the major draining lymph node. Efficiency of
immunization was also dependent on the promoter and
expression vector used. The cytomegalus virus promoter
driven pCMV␤ vector was superior to the Moloney murine
leukemia virus LTR driven BAG vector. LacZ DNA immunization was also compared with cell-based vaccination with
lacZ-transfected tumor cells, in which case again the pinna
was the best site for inducing strong immune responses.
Tumor-specific T cell responses could also be well induced
in the pinna, leading to cytotoxic T lymphocyte induction
and protective antitumor immunity. Thus, the pinna was
found to be a privileged site for induction of antitumor
responses and for genetic immunization, an important finding of immediate practical and potential future clinical
implications.
Keywords: lacZ gene; promoter; tumor; immunity; genetic immunization
Introduction
In 1990, Wolff and colleagues1 reported that nonreplicating DNA plasmids encoding reporter genes could be
internalized and the encoded proteins expressed by
muscle cells following injection of plasmids directly into
muscle without the use of any transfection vehicle. The
subsequent finding that naked plasmid DNA encoding a
foreign protein could also be immunogenic upon intramuscular inoculation opened an entirely new area of
research with great clinical impact. Somatic gene therapy
is becoming increasingly important for potential treatment of genetic and acquired disorders. Without any specific delivery system the direct DNA transfer into tissues
is followed by uptake of plasmid molecules into the cytoplasm and subsequently into the nucleus of cells. They
are then translated into functional proteins, processed
into peptides and presented by major histocompatibility
(MHC) molecules to immune T cells.2 Direct DNA transfer has been successfully used for in vitro induction of
both humoral and cellular immune responses to several
antigens, including viruses such as influenza, hepatitis B,
or HIV (human immunodeficiency virus), parasites, or
tumor antigens.3–8 Additionally, systemic effects were
observed after transfer of cytokine genes.9
Correspondence: V Schirrmacher
Present addresses: 2Pharmacia and Upjohn GmbH, 91058 Erlangen, Germany; and 3SmithKline Beecham Biologicals, Rue de l’Institut 89, B-1330
Rixensart, Belgium
Received 8 August 1997; accepted 18 December 1997
It was assumed at the beginning that striated muscle
is the only tissue in vitro suitable to take up and express
naked DNA.10 Soon, however, it turned out that other
organs showed a similar ability for expression of a
reporter gene.11 The skin as a possible target tissue for
genetic immunization was successfully tested by Tang et
al,12 by bombardment with gold microprojectiles coated
with plasmid DNA. But gene expression in the skin was
also observed when using needle injection of plasmid
DNA,13,14 especially when applying high-frequency puncturing of the skin.15 For effectiveness of gene therapy the
skin seems to be superior to muscle or other organs.
Muscle is not considered to be a site specialized for antigen presentation because it contains few if any antigen
presenting cells (APCs) or lymphocytes. Thus, bone marrow-derived nonmuscle cells were shown to be involved
in the induction of cytotoxic T lymphocytes (CTL) following intramuscular DNA immunization.16–18 In contrast,
the skin and mucous membranes are the physiological
sites where most exogenous antigens are normally
encountered. The skin-associated lymphoid tissues contain specialized cells such as keratinocytes, macrophages
and Langerhans’ cells, which are involved in the
initiation and further augmentation of immune
responses. Langerhans’ cells carry the antigen from the
skin to draining lymph nodes where they function as professional APCs for priming naive T lymphocytes. The
transfection of skin cells by DNA is therefore expected to
be an efficient route of immunization, mimicking a physiologic response as during infection.
Taking advantage of the reporter gene lacZ which can
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P Forg et al
790
easily be monitored we compared the ability of skeletal
muscle and different sites of skin to take up and express
naked lacZ DNA and to induce antibody and CTL
responses against the encoded model antigen ␤-galactosidase (␤-gal). In previous studies with the ESb tumor
system it was shown that the ear pinna is a privileged
site for the induction of antitumor immunity,19 preventing the outgrowth of an otherwise lethal dose of
tumor cells. Thus it was reasonable to test the ear pinna
also as a putative site for DNA vaccination and to compare the strength of the immune response to that induced
by ␤-gal expressing tumor cells. Additionally, two different lacZ encoding vectors were compared for usage as
vaccines. Here we demonstrate that the strongest
immune responses to DNA vaccination were obtained
after intra ear pinna (i.e.) and not after muscle immunization and that the immune response intensity did not
correlate with the duration of gene expression in the
injected tissues.
Results
Comparison of expression of lacZ DNA in skeletal
muscle, abdominal skin and ear pinna after direct DNA
transfer
We first compared different tissues for their capability of
taking up injected lacZ DNA and expressing the foreign
gene over a certain period of time. Two different vectors
were used: the retroviral vector BAG and the expression
vector pCMV␤. Following inoculation, animals were
killed at different time-points, and their frozen tissue sections stained for ␤-gal-positive cells ex vivo (Figure 1a–f).
With both lacZ encoding vectors a direct transfection was
observed. Especially in the case of the intradermal injections at the abdominal skin or the ear pinna, the stained
area appears to represent the direction of the needle during the prick (Figure 1b and c). An analysis of the ear
pinna by X-gal-stained histological cross-sections (Figure
1e) revealed that many cell types within the dermis and
epidermis took up and expressed the DNA without
any preference.
Expression was seen longer in muscle tissue (Figure 1a)
(quadriceps at least for 4 weeks) than in the abdominal
skin (Figure 1b) or the ear pinna (Figure 1c–e), but it was
also dependent on the vector used. Expression vanished
earlier with pCMV␤ than with BAG. Three weeks after
pinna injection, no blue cells could be detected. The kinetics of gene expression were studied in more detail for
the ear pinna. Using pCMV␤, distinct expression as a
blue spotted circular area had already occurred after 1
day. This characteristic pattern changed to a few lines
(Figure 1d) within 10 days. After 14–21 days, in most
cases, no more expression was visible. A similar gradual
decline of gene expression over time was seen in nude
mice. Figure 2 illustrates the relative strength and duration of ␤-gal gene expression at the different injection
sites.
Detection of ␤-galactosidase-positive cells in the
draining lymph nodes after i.e. injection
Analysis of the draining lymph nodes (dLn) after i.e.
DNA injection revealed the presence of ␤-gal-positive
cells (Figure 1f). In control lymph nodes of nontreated
ear pinnae such X-gal staining of cryosections was never
a
b
c
d
e
f
Figure 1 Expression of ␤-gal following injection of DNA into different
murine tissues. (a) Whole organ staining of the quadriceps of the left hind
leg ex vivo 30 days after injection of BAG. (b) Skin from the shaved flank
21 days after injection of BAG. (c and d) Ventral site of an ear pinna,
injected with pCMV␤ 1 day (c) and 10 days (d) before. (e) Tangential
section of an ear pinna with ␤-gal expressing cells (× 20). (f) A ␤-galpositive cell in a cryosection of the draining lymph node detected by Xgal staining 3 days after injection of pCMV␤ into the corresponding ear
pinna (× 100).
Figure 2 Kinetics of ␤-gal expression after pCMV␤ lacZ DNA inoculation into the ear pinna (i.e.), into the dermis of the flank (i.d.) or into
muscle tissue (i.m.). Expression strength is expressed by an arbitrary scale
of 1 (low), 2 (medium) and 3 (high). Early time-points (3–24 h) were only
tested i.e. Expression duration was i.m. ⬎ i.d. ⬎ i.e.
observed. The first time-point after i.e. pCMV␤ inoculation when blue cells could be detected in the major dLn
(superfiscia cervicalis) was at 1 day. The type of cell in the
lymph node which expressed ␤-gal could not be clearly
identified. When the pinna was removed 30–60 min after
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P Forg et al
injection, no blue cells were detectable at any time later
in the dLn. This differed from a situation, in which lacZtransfected tumor cells were i.e. inoculated and in which
stained cells could be seen in the dLn as early as 15 min
after inoculation.20
Induction of ␤-gal-specific antibodies
The development of humoral immunity, in the form of
specific IgG anti-␤-gal antibodies, was tested using a
sensitive ␤-gal ELISA plate assay. Serum titers are given
as relative values in comparison to a standard monoclonal anti-␤-gal antibody at a concentration of 10−5
mg/ml. The efficacy of the different sites of injection with
regard to induction of humoral immunity are compared
in Figure 3. Independent of the injection site, a single
inoculation of BAG induced distinct antibody levels.
These were detectable after 2 weeks and increased constantly over the observed period of 12 weeks. While
inoculation into the quadriceps (Figure 3a) or into the
abdominal skin (Figure 3b) revealed a similar antibody
response, i.e. immunization (Figure 3c) resulted in a
much stronger response which reached a plateau after 4
weeks. Sera from mice immunized i.e. had to be diluted
much further to reveal the whole titration curve.
Inoculation of live ESbL-lacZ tumor cells into the ear
pinna also resulted in the production of specific anti-␤-
gal antibodies. In comparison with DNA-based vaccination, the antibody increase was more rapid (Figure 3d).
Induction of a MHC class I restricted ␤-gal peptidespecific CTL response
To evaluate the development of a T cell-mediated
immune response, splenocytes from mice immunized
with BAG or pCMV␤ intramuscular (i.m.), intradermal
(i.d.) or i.e., were used to set up a CTL restimulation culture in the presence of a known Ld-restricted peptide of
the dominant ␤-gal-derived T cell epitope. As target cells
for the subsequent 4h 51Cr release assay the lacZ expressing P13.1 cells were used and as negative control the
respective nontransfected parental P815 mastocytoma
cells. Spontaneous release was always below 10%. Results
of representative experiments are shown in Figure 4.
After i.m. immunization with BAG for 9–21 days no specific CTL responses could be generated (Figure 4a). In
contrast, after i.d. immunization (Figure 4b) distinct
responses were reproducibly observed with around 50%
lysis at 50:1 effector to target cell ratio, when the immune
spleen cells were used on day 21. The cytolytic activity
generated in these bulk cultures was even higher when
BAG immunization was performed i.e. (60–70% specific
lysis, Figure 4c). Priming times with BAG shorter than
3 weeks was not sufficient for induction of strong CTL
Figure 3 Kinetics of humoral anti-␤-gal immune responses. Serum samples from three animals per group were tested in triplicate on ␤-gal ELISA
plates. The average values were calculated over the OD492 of monoclonal anti-␤-gal at a concentration of 10−5 mg/ml. Single immunization with BAG
i.m. (a), i.d. (b) and i.e. (c). (d) Cell-based immunization with ESbL-lacZ tumor cells.
791
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792
with ESbL-lacZ tumor cells, however, can be totally
addressed to the effect of ␤-gal since restimulation with
the peptide is ␤-gal specific.
Figure 4 ␤-gal peptide-specific CTL responses in lacZ-immunized mice.
Splenocytes from three mice per group were isolated 3 weeks after a single
injection of the lacZ vector BAG i.m. (a), i.d. flank (b), and i.e. (c) or 9
days after i.e. injection of ESbL-lacZ cells (d) or of the lacZ vector pCMV␤
(e). The immune spleen cells were restimulated in bulk cultures for 5
days with 0.5 ␮g/ml TPHPARIGL peptide. The thus generated Ld-peptiderestricted CTL effector cells were then tested in a 4 h 51Cr release assay
against P13.1 cells expressing ␤-gal (쮿), or against the parental P815
cells (쑗) as negative control. (f) Effect of resection of the DNA-injected
ear pinna at different time-points after immunization. Mice were immunized as in (e). In separate groups of three mice each the pinna was resected
either 1 h (왔), 24 h (쏔) or 72 h (쏆) after DNA inoculation. Effector
cells were generated as in (e) and tested against P13.1 target cells.
responses (data not shown). The strongest ␤-gal-specific
CTL response was observed after i.e. immunization with
live lacZ-transfected ESbL lymphoma cells (Figure 4d). In
addition to the strong immune response to the foreign
lacZ gene product, strong ESb tumor-specific CTL
immune responses are induced at this injection site.21 The
observed CTL response (Figure 4d) after immunization
Promoter-dependent differences in the induction of
immune responses and relevance of antigen
persistence
When pCMV␤ was used instead of BAG, a quicker and
stronger ␤-gal peptide-specific CTL response was
observed. Thus, 30% specific lysis could be generated
from immune spleen cells of mice primed for only 9 days
by i.e. immunization (Figure 4e). Removal of the injected
ear pinna at different time-points after pCMV␤ injection
revealed that the strength of the induced CTL responses
increased with the time of pinna persistence. No CTL
response was seen when the pinna was removed 1 h after
DNA application, while weak responses were seen when
the injected pinna was allowed to persist for 24 or 72 h
(Figure 4e). Even a 72 h lasting persistence did not reach
the same CTL capacity as a persistence for the whole
priming time of 9 days. Thus, persistence of the DNAinjected pinna over a certain period of time (⬎72 h)
appeared as a prerequisite for obtaining optimal CTL
responses.
When anti-␤-gal immune responses were compared at
the same time-point after i.e. injection of the two different
lacZ expression vectors, stronger humoral (Figure 5a) and
T cell-mediated responses (Figure 5b) were observed
after immunization with pCMV␤ than with BAG.
A kinetic analysis of the pCMV␤-induced i.e. response
revealed that good CTL responses were already obtainable after 6 days of DNA immunization. The lytic activity
remained high (approximately 80% specific lysis) for at
least 45 days (data not shown).
Another point of interest was the influence of B and T
cell-mediated immune responses of a second DNA injection. To test this, 14 days after the first i.e. injection of
pCMV␤ mice received the same dose of pCMV␤ at the
same site. As shown in Figure 5a, this boosting injection
increased the antibody titer further. Similarly, a boosting
injection of pCMV␤ and a total priming time of 28 days
led to a further increase in the peptide-generated CTL
activity (Figure 5b). The lysis was antigen specific since
nonimmunized mice as well as mice immunized with a
nonrelevant plasmid which did not encode the lacZ gene
displayed only background activity (data not shown).
In summary, we demonstrate important parameters for
optimal immune responses after genetic immunization
with a model antigen: the site of injection, the promoter
used and the number of injections performed.
Mouse pinna as a privileged site for induction of
antitumor responses
The pinna was also a privileged site for induction of antitumor immune responses. Results obtained with the welldefined DBA/2 mouse lymphoma ESb which expresses
a tumor characteristic Kd-restricted CTL epitope22 are
shown in Table 1. While inoculation of 5 × 104 live tumor
cells at s.c., i.m. or i.d. (flank) sites (groups I–III) led to
progressive tumor growth and death from metastases
within 2–3 weeks, i.e. inoculation (group IV) induced
tumor resistance and specific protective antitumor
immunity as revealed by tumor re-challenge experiments. Induction of protective immunity by i.e. tumor
immunization was abrogated by sublethal irradiation
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P Forg et al
(group VII) or pretreatment with depleting anti-CD4 (V)
or anti-CD8 (VI) monoclonal antibodies (mAbs). ESbspecific CTL responses could be induced by i.e. priming
and intraperitoneal (i.p.) restimulation (groups VIII–X).
The peritoneal exudate cells mediated syngeneic antitumor cytotoxicity and lysed ESb, but not Eb lymphoma
cells which express a different tumor-associated antigen
(group VIII). Furthermore, the ESb-specific kill could be
blocked by anti-MHC class I Kd but not anti-Dd mAbs,
thus revealing the restricting molecule for the tumorspecific CTL as Kd.
Discussion
In order to optimize DNA vaccination protocols, we
evaluated the efficacy of gene transfer in several tissues
and followed subsequent immune responses to ␤-gal as
model antigen. Mice were inoculated into skeletal muscle, skin and ear pinna with two different vectors containing the bacterial lacZ gene under the transcriptional control of the Moloney murine leukemia virus LTR or the
CMV promoter. The read out systems for the study were
the examination of the lacZ gene expression, the production of anti-␤-gal antibodies and the priming for ␤gal peptide-specific CTL responses.
In situ staining by X-gal revealed that all three tissues
were able to take up and express the plasmid DNA. No
differences were detected between the two vectors BAG
and pCMV␤ with regard to the quantity of lacZ mRNA
in injected tissues (data not shown) and to the level of
gene expression in situ. The skeletal muscle expressed ␤gal in a broad area after injection of lacZ encoding vectors
(Figure 1a), whereas the stained cells of skin and ear
pinna were arranged around a well defined region
(Figure 1b–d) which seems to reflect the direction of the
needle during inoculation. Cross-sections of the ear pinna
(Figure 1e) displayed distribution of ␤-gal expressing
cells throughout the whole organ, including the epidermis and dermis. The alteration of expression strength and
pattern noticed in the ear pinna over time is in agreement
with observations made by Hengge et al23 in pig skin,
with a decline of expression after 3 weeks and a gradual
reduction in size of the transfected stretch. One possibility could be that the decline of expression was due to
an immunological process as was described in muscle
where an inflammatory reaction occurred in response to
the injection of naked DNA.24 This seems, however
unlikely since we found the same decline of expression in
the ear pinna of immune-deficient nude mice. Moreover,
inflammatory reactions of the skin were not observed in
this study. Another possibility is down-regulation of promoter activity. Transient expression of transgenes under
the control of the CMV promoter was observed by other
investigators.14,25 For clinical applications it may be considered an advantage when expression of a foreign gene
is transient and is switched off after induction of immunity. It might be safer and easier to control. Immunizations
could be repeated if necessary.
Muscles as well as skin have already been described
as targets for DNA transfer. The skin is an especially
interesting tissue since it carries a variety of cells that
could induce or be involved with immune responses, eg
keratinocytes, macrophages or Langerhans’ cells. The
thin ear pinna exhibits two layers of epidermis and dermis, separated by cartilage, thereby doubling the amount
of professional APC. So far, the ear pinna tissue as a special site and form of the skin has not been investigated
in gene therapy in detail although its anatomical properties and immunological features seem to predestine it for
this purpose. It is easily accessible without the need for
pre-treatments such as shaving, and has been demonstrated to be a privileged organ for the induction of protective antitumor immune responses.19,20,26
An interesting point is the detection of ␤-gal expressing
cells in the draining lymph node after i.e. injection of
lacZ. We could not see such blue stained cells when the
injected ear pinna was removed after 1 h. Our assumption is that these stained cells represent Langerhans’ cells
or dermal dendritic cells which have been transfected
directly and then migrated into the draining lymph node
Figure 5 Comparison of i.e.-induced immune responses to different lacZ plasmid expression vectors. Humoral (a) and cell-mediated (b) anti-␤-gal
immune responses after a single i.e. immunization on day 0 with BAG (쮿) or pCMV␤ (쑗) or after two i.e. immunizations on day 0 and 14 with
pCMV␤ (쎲). (a) Serum samples from three animals per group taken before (*) or 4 weeks after immunization were ELISA tested as in Figure 3. (b)
␤-gal Ld peptide-restricted CTL responses were assayed as in Figure 4 with day 28 immune spleen cells against P13.1 target cells (*: lysis of P815
control cells).
793
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794
Table 1 Mouse pinna as a privileged site for induction of antitumor responses
Group
I
II
III
IV
V
VI
VII
ESb tumor cell
inoculuma
Site
Additional treatmentb
% ESb tumor resistanced
5 × 104
5 × 104
5 × 104
5 × 104
5 × 104
5 × 104
5 × 104
s.c.
i.m.
i.d.
i.e.
i.d.
i.e.
i.e.
—
—
—
—
d-1 ␣ CD4
d-1 ␣ CD8
d-1 5 Gy
0
0
0
80e
0
0
0
Restimulation
% Specific cytotoxicity against
ESb
Eb
Priming
VIII
IX
X
5 × 104
5 × 104
5 × 104
i.e.
i.e.
i.e.
ESb irr. d-12 i.p.c + medium
ESb irr. d-12 i.p. + ␣Kd
ESb irr. d-12 i.p. + ␣Dd
55.1
9.8
57.0
0.5
1.5
2.0
a
DBA/2 mice were immunized with live syngeneic ESb 289 lymphoma cells on day 0 (d0) at the sites indicated.
Separate groups of mice (10 per group) were pretreated i.p. 1 day before with 250 ␮l ascites fluid of either anti-CD4 (GK 1.5) or antiCD8 (YTS) mAb or were pretreated by 5 Gy sublethal X-irradiation.
c
For generation of tumor-specific CTL activity, mice were restimulated with 1.5 × 107 X-irradiated (100 Gy) ESb tumor cells i.p. for 3
days and the harvested peritoneal effector cells (PEC) further cocultured for 3 days with ESb stimulator cells. 51Cr-labeled ESb target
cells (which express a Kd-restricted tumor-specific CTL epitope) or Eb target cells (which express a different tumor-associated antigen)
were preincubated for 45 min at 4°C with either medium or five-fold concentrated hybridoma supernatants before PEC effector cells
were added in a 2.5-fold excess to the targets and incubated for 4 h.
d
Represents the per cent survivors after 60 days. Mortality occurred between days 8 and 18 and was due to metastases.
e
These mice developed specific protective antitumor immunity so that after re-challenge i.d. with either Eb or ESb lymphoma cells Eb
but not ESb tumors developed.
b
to function as APCs. We cannot exclude the possibility
that these cells might have taken up the protein from
dying cells and have then migrated into the lymph node
to present the antigen to lymphocytes but it is not likely
that the ␤-gal would then be in enzymatically active form
that allows detection by X-gal staining. The same
phenomenon, the presence of foreign DNA expressing
dendritic cells in draining lymph nodes after cutaneous
DNA-based immunization was recently reported by Condon et al.27 Other studies pointed towards an important
role of host APC in the induction of T cell-mediated
immune responses after i.m. DNA injection. Such APC
did not need to be transfected themselves.16–18
The presence of the foreign antigen ␤-gal in the lymph
nodes was a first hint for induction and generation of an
immune response. The generation of a specific humoral
immune response against the encoded protein after genetic immunization was seen for every site of injection, but
the titers were influenced by the injection site. Intramuscular and intradermal immunizations exhibited a similar
development with a rather low antibody titer. Animals
injected with DNA i.e. displayed an IgG antibody level
after 2 weeks that was similar to that observed as maximal after i.m. or i.d. injections. This may reflect the
strength of MHC class II-mediated helper T cell immunity induced by i.e. immunizations. A further substantial
antibody response increase was demonstrated after
injecting pCMV␤ a second time 2 weeks after the initial
DNA transfer. This is in contrast to Ertl and Xiang25 who
saw no boosting effect and proposed the augmentation
to be the result of a slow on-rate. Comparison of the titers
of antibody induced after inoculation of BAG or pCMV␤,
respectively, revealed the CMV promoter-driven
expression to be more efficient. This may reflect a specific
activity of the CMV promoter, confirming earlier
results,28 and probably demonstrates the connection
between level of expression and strength of the generated
immune response.
A similar conclusion concerning antigen dose dependency of the response can also be drawn from immunizations with ␤-gal expressing tumor cells. Intra-pinna
injection of a subtumorigenic dose of ESbL-lacZ as well
as of ESb cells (Table 1) led to induction of antitumor
immunity so that a subsequent tumor challenge at a
tumorigenic site was rejected by syngeneic DBA/2 mice.
This effect was dependent on CD4 and CD8 T cells since
injection of depleting anti-CD4 or anti-CD8 mAbs would
prevent induction of immunity against the ESbassociated tumor antigens (Table 1 and Ref. 29). The
additional foreign protein ␤-gal in ESbL-lacZ could
induce additional anti-␤-gal T cell responses (Figure 3d)
thereby increasing the immunogenicity of the tumor
cells.21 The production of anti-␤-gal antibodies after
tumor cell-based immunization was higher than after
genetic immunization. After DNA injection, the number
of cells expressing ␤-gal is presumably lower than the
5 × 104 ESbL-lacZ tumor cells injected i.e. The induced
anti-␤-gal immune responses could also confer protective
immunity against other ␤-gal-transfected tumor cells
(unpublished observations).
Differences as seen with the humoral immune
responses were also observed when CTL-mediated
peptide-specific immune responses were tested. Muscle
cells injected with BAG totally failed to induce MHC
class I-(Ld) ␤-gal peptide-restricted CTL activity, while
after intradermal immunizations good CTL responses
were elicted. Thus, while humoral responses were equivalent for i.m. and i.d. injections, induction of cytotoxic
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P Forg et al
CD8+ T cell precursors differed distinctly between these
two tissues. Reasons for this may be seen in the different
APC content of the injection sites in combination with the
rather low level of expression. A weak CTL response
could be elicited i.m. when the stronger pCMV␤ vector
¨
was applied (data not shown). Bohm et al,30 however,
presented data in their system suggesting that lower
expression stimulates only MHC class I-mediated T cell
responses while a higher expression resulted in both
humoral and cellular immunity. In contrast, in our system, the lower level of expression after i.m. injection of
BAG seemed to induce preferably B cell-mediated
responses.
With regard to the peptide-specific CD8 T cellmediated CTL response, the intra-pinna inoculation site
proved again to be superior to muscle and abdominal
skin. The induced CTL response was nearly comparable
to the response after injection of ESbL-lacZ cells so that
in this case the amount of antigen was not as decisive as
for the humoral responses. Again, a booster injection
could further augment the CTL immune response. A
main difference between the genetic and the cell-based
immunization in the ear pinna relates to the effect of
removal of the injected pinna at different time-points
after injection. For direct DNA transfer we demonstrated
the need for persistence of the pinna, possibly as an antigen reservoir, during the priming period for the development of a strong CTL response. Previously, we showed
in a similar experimental set-up concerning tumor cellbased immunization in the ear pinna that the cytotoxic
antitumor immune response is generated at comparable
levels even if the pinna is removed after 1 h.20 In the case
of i.e.-injected lacZ-tumor cells, lacZ-labeled cells could
be detected in the draining lymph node and spleen as
early as 15 min after injection,20 while in the case of i.e.injected lacZ DNA, at least 24 h were required before
lacZ-labeled cells could be detected in the draining node.
Thus, although the strength of immune responses
induced either by DNA or cell-based immunization may
be similar, the kinetics of induction and the mechanism
of priming seem to be quite different.
In summary, the ear pinna was demonstrated to be an
optimal site for generation of humoral and cell-mediated
immune responses after DNA vaccination. One reason
for this may be the local and focal concentration of antigen in a restricted area connected with the one major
draining lymph node (superfiscia cervicalis). This can
result in fast stimulation of virgin T lymphocytes by
antigen-loaded dendritic cells. After i.d. or i.m. injection,
the response is likely distributed over several lymph
nodes.31 The mechanical irritation by needle injection
may induce local cytokine secretion and activate and
recruit additional APC.
Regarding the route of DNA administration, the
experiences of different groups differ widely. Donnelly et
al32 reported that i.m. treatment resulted in a better protection than i.d. application. There are hints33 that gene
gun-mediated immunization gives similar effects as i.d.
injections with predominantly Th2 immune responses,
but with similar CTL activity as after i.m. injection. However, recently, i.d. injection of the lacZ gene was found
to preferentially induce Th1 response.34 In accordance
with this, we demonstrated recently a superiority of the
i.e. over a s.c. tumor inoculation site for induction of a
Th1 type cytokine response.26 Fynan et al35 observed that
i.m. applications were weaker in elicting immune
responses than gene gun treatment. Our results show that
i.d. and especially i.e. injections, are the most efficient
ways of inducing strong immune responses. In these situations, the entrance of the antigen via the skin may be
efficient because it follows routes of natural infection. The
type of antigen may also affect the type of immune
response generated. However, the route of administration seems to have greater influence, pointing towards
outstanding potential of the ear pinna as a DNA vaccination site.
Materials and methods
DNA
The retrovirus-based vector BAG (approximately 11 kb)36
was obtained from Dr C Cepko (Dept. of Genetics, Harvard Medical School, Boston, MA, USA), and the
expression vector pCMV␤ (7, 2 kb) from Clontech
(Heidelberg, Germany, Cat No. 6177-1; GenBank
Accession No.: U02451). The expression of the lacZ gene
is driven by the Moloney murine leukemia virus LTR
promoter in the BAG vector, which also contains a neomycin resistance gene under the control of a SV40 promoter. In the pCMV␤ vector lacZ expression is regulated
by the CMV immediate–early gene promoter. Plasmids
were grown in Escherichia coli and purified using the
Qiagen Mega Prep Kit (Qiagen, Hilden, Germany). The
DNA, with a 260:280 ratio from 1.8 to 2.0, was dissolved
in water for storing. For in vitro injections, the DNA was
freshly diluted and adjusted to 1 ␮g/␮l in 1 × PBS final
concentration.
Mice
Pathogen-free female DBA/2 mice were purchased from
IFFA Credo (Lyon, France) and used at 6–12 weeks of
age.
Cell lines
ESbL-lacZ, a highly metastatic lacZ-transfected lymphoma,28 P815, a mastocytoma, and its lacZ-transfected
variant P13.137 were cultivated in RPMI-1640 medium, as
described earlier.38
Peptide
The synthetic nonamer TPHPARIGL represents the
naturally processed H-2Ld-restricted epitope of ␤-gal
spanning amino acids 876–884.39 It was synthesized by R
Pipkorn (DKFZ).
Immunizations with DNA and tumor cells
Fifty micrograms of DNA, dissolved in PBS, were
injected either i.m. or i.d. into the shaved flank by using
27-gauge needles, or into the ear pinna of anesthetized
animals using 32-guage needles. For i.m. injections a volume of 100 ␮l was used, whereas for i.d. and i.e. 50 ␮l
volumes were administered. Anesthesia was carried out
by i.p. injection of Rompun- (Parke Davis, Berlin,
¨
Germany) Ketanest- (Bayer, Leverkusen, Germany) PBS
(1:1:3). For immunization with tumor cells, a subtumorigenic dose of 5 × 104 ESb or ESbL-lacZ cells in 50 ␮l PBS
was injected i.e.
ELISA
Blood samples were obtained from the tail vein of mice
every second week. Sera were prepared and stored at
795
Intra-pinna DNA vaccination
¨
P Forg et al
796
−20°C until they were analyzed for anti-␤-gal antibodies
in ELISA. For ELISA, 96-well plates were coated overnight with purified ␤-gal (Sigma, Deisenhofen, Germany)
in 0.05 m carbonate buffer at 4°C. After washing with
0.05% Tween-20/PBS the plates were blocked with 0.2%
gelatine in PBS for 1 h at 37°C. Sera were serially diluted
in Tween/PBS and applied to the plates for 1 h at room
temperature. After further washing steps, bound antibodies were detected with a peroxidase-conjugated goat
anti-mouse IgG immunoglobulin (Dianova, Hamburg,
Germany). The plates were washed and finally developed
with ortho-phenyl-diamine substrate buffer and read at
OD 492 nm. As a control, a monoclonal mouse anti-␤-gal
antibody (Boehringer Mannheim, Mannheim, Germany)
was applied in different concentrations.
Effector cells
Animals were immunized with 50 ␮g lacZ DNA i.m., i.d.
or i.e., respectively. Nine to 28 days later, 2 × 107 spleen
cells were restimulated in vitro for 5 days in RPMI
medium containing 10% FCS and 0.5 ␮g/ml TPHPARIGL. For the cell-based induction of lacZ immunity,
5 × 104 live ESbL-lacZ in PBS were inoculated i.e. and
splenocytes were restimulated in vitro after 9 days.
51
Cr release assay
The effector cells were tested for their cytotoxic activity
in a standard 4 h 51Cr release assay at different
effector:target ratios against 5 × 103 51Cr-labeled P13.1
and P815 cells, respectively, as described earlier.21,38 The
amount of 51Cr released was measured in a gamma counter and the percentage of lysis was calculated from
the formula: [(experimental cpm − spontaneous cpm)/
(maximum cpm − spontaneous cpm)] × 100.
X-Gal staining
Organs were removed from the animals and stained
immediately by washing in PBS and incubating in fixation solution (2% formaldehyde/0.2% glutaraldehyde in
PBS) on ice for 1 h. For this purpose, ear pinnae were
peeled to separate the dorsal and ventral sides. After
washing in PBS, organs were incubated in X-gal solution
(5 mm K3Fe(CN)6, 5 mm K4Fe(CN)6, X 3 H2O), 2 mm
MgCl2, 0.01% sodiumdesoxycholate, 0.02% NP40, 1 mg/
ml X-gal (Boehringer Mannheim) at 37°C for 3–16 h.
Lymph nodes were snap-frozen in liquid nitrogen and
cut with a cryostat (Leica, Bensheim, Germany), where
on average every sixth piece was taken for analysis. After
fixation for 10 min on ice and washing in PBS the slices
were stained in X-gal solution as described.
Acknowledgements
The financial support of SmithKline Beecham Biologicals
¨
for Petra Forg is gratefully acknowledged.
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