Dendritic cells presenting equine herpesvirus

Journal
of General Virology (1998), 79, 3005–3014. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Dendritic cells presenting equine herpesvirus-1 antigens
induce protective anti-viral immunity
Falko Steinbach,1 Kerstin Borchers,1 Paola Ricciardi-Castagnoli,2 Hanns Ludwig,1 Georg Stingl3
and Adelheid Elbe-Bu$ rger3
Institute of Virology, FU Berlin, Ko$ nigin-Luise-Str. 49, 14 195 Berlin, Germany
Department of Pharmacology, CNR Centre of Cytopharmacology, University of Milan, Via Vanvitelli 32, 20 129 Milan, Italy
3
DIAID, Department of Dermatology, University of Vienna Medical School, VIRCC, Brunner Str. 59, 1235 Vienna, Austria
1
2
Equine herpesvirus-1 (EHV-1) causes rhinopneumonitis, abortion and CNS disorders in horses.
Using intranasal inoculation, the mouse model of
this disease mimics the major pathogenic and
clinical features of the equine disease. The aim of
this study was to investigate whether murine dendritic cells (DC) can be infected with EHV-1 and
whether they can be used as cellular vaccines for the
induction of prophylactic anti-EHV-1 immunity. It
was found that the DC lines FSDC, D2SC1, 18 (all
H-2d) and 80/1 (H-2k), when incubated with the Ab4
strain of EHV-1, do not change their morphology
and phenotype but sustain virus replication and
induce proliferation of naive, syngeneic T cells. An
even stronger proliferation of T cells was seen when
DC were used that had been pre-exposed to heat-
Introduction
Equine herpesvirus-1 (EHV-1) is an important pathogen of
horse populations worldwide and is responsible for a variety of
clinical problems including respiratory distress, abortion and
neurological disorders (for references Crabb & Studdert, 1995).
Using intranasal inoculation, a mouse model was established a
few years ago, and the pathogenesis of the infection in these
mice has features that resemble the natural infection in horses,
including a transient viraemia and a pronounced rhinopneumonitis (Awan et al., 1990 ; Inazu et al., 1993). After
primary infection, alphaherpesviruses (α-Herpesvirinae) are
known to remain latent at neuronal sites. Considering the
recent detection of EHV-1 in trigeminal ganglia as one site of
latency in specific pathogen-free horses and mice (Slater et al.,
Author for correspondence : Falko Steinbach.
Fax ­49 30 831 6198. e-mail fstvirol!zedat.fu-berlin.de
0001-5721 # 1998 SGM
inactivated virus. DC lines were therefore pulsed
with inactivated virus and were then administered
intranasally to either BALB/c or C3H mice on days
N25, N15 and N5. Control groups received either
medium, unpulsed DC or inactivated virus only.
Animals were challenged with EHV-1. Whereas mice
of control panels showed clinical signs of EHV-1
disease and 27 % died, animals immunized with the
pulsed DC lines showed only subtle clinical
symptoms, lost significantly less weight, exhibited a
reduced virus load in their lungs and CNS and did
not succumb to the disease during the observation
period. These results show that murine DC can
present EHV-1 and initiate a protective anti-viral
immunity in vivo.
1994 b ; Baxi et al., 1996), EHV-1 resembles a classical αherpesvirus and the mouse model seemed to be a suitable tool
to study generalized infections with α-Herpesvirinae in vivo.
In horses and mice, the anti-viral immunity to EHV-1
consists of neutralizing antibodies, natural killing mechanisms
and cytotoxic T lymphocytes (CTL). The latter cells are
thought to play a key role in protection (Chong et al., 1992 ;
Chong & Duffus 1992 ; Alber et al., 1995 ; Allen et al., 1995 ;
Edens et al., 1996), but the exact conditions for most effectively
inducing such a protective response are still a matter of debate.
When administered in relatively large amounts, live EHV-1, as
well as recombinant glycoproteins gB, gC and gD, can confer
protection (Tewari et al., 1994, 1995 ; Osterrieder et al., 1995 ;
Stokes et al., 1996). Adoptive transfer of T lymphocytes has
successfully been used to reduce virus replication and provide
protection in mice (Azmi & Field, 1993 a, b), but it remains
unclear which precise role CD4+ and CD8+ T lymphocytes
play in the initiation of the protection.
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F. Steinbach and others
et al., 1995 ; Bo$ hm et al., 1995 ; Elbe & Stingl, 1995 ; Majordomo
et al., 1995 ; Bachmann et al., 1996 ; Hsu et al., 1996 ; Porgador
et al., 1996 ; Smith et al., 1996 ; Young & Inaba, 1996). Thus far
and to our knowledge, the use of DC to induce anti-viral
immunity in vivo has not been reported. The different methods
used to obtain DC, mainly from bone marrow, blood and skin,
do not always result in fully homogeneous and}or comparable
populations. To circumvent this problem, we took advantage
of previously established stable, long-term DC lines from
mouse skin and spleen (Paglia et al., 1993 ; Elbe et al., 1994 ;
Girolomoni et al., 1995). With the aim of developing more
broadly active and efficient strategies for the induction of
prophylactic anti-EHV-1 immunity, we investigated whether
DC can stimulate T cell immunity to EHV-1. We report here
that EHV-1 infects several murine DC lines productively and
that DC pulsed with heat-inactivated virus are able to stimulate
naive, syngeneic CD8+ T cells in vitro, and initiate protective
immunity against this virus in vivo.
Methods
+ Animals. BALB}c (H-2d) and C3H}HeJ (C3H) (H-2k) inbred mice
(3–4 weeks old) were obtained from the Dept of Animal Breeding, Robert
von Ostertag Institute Berlin, Germany and Bomholtga/ rd, Bomholtvej,
Denmark, respectively.
+ Cell lines. The establishment and culture conditions for the DC lines
FSDC, D2SC1, DC18 (all H-2d) and 80}1 DC (H-2k) have been described
elsewhere (Paglia et al., 1993 ; Elbe et al., 1994 ; Girolomoni et al., 1995).
The fibrosarcoma cell line L929 (H-2k, CCL1) and the monocyte}
macrophage cell line J774A.1 (H-2d, TIB67) were obtained from the
ATCC. The mouse keratinocyte cell line PAM212 (H-2d) (Yuspa et al.,
1980) was maintained in normal cell culture medium (NCM ; RPMI 1640
supplemented with 10 % heat-inactivated FCS, 2 mM -glutamine, 1 mM
sodium pyruvate, 0±1 mM non-essential amino acids, 25 mM HEPES,
50 µg}ml gentamycin, 1¬ antibiotic}antimycotic solution and 50 µM 2mercaptoethanol, all from GIBCO Life Technologies. The rabbit kidney
cell line RK-13 (ATCC, CCL37) and the equine dermis cell line ED
(ATCC, CCL57), used for propagation of virus, were kindly provided by
H. Field, Cambridge, UK and P. Thein, Munich, Germany, respectively.
Fig. 1. Replication of EHV-1 in selected cell lines. (A) 104 DC were
incubated for 2 h with 104 p.f.u. EHV-1 strain AB4 and cultured for 5 days
in 1 ml culture medium. The amount of virus that can be rescued from
infected DC was monitored daily by co-cultivating an aliquot thereof with
RK-13 cells. The amount of virus per DC is presented in (B). All DC lines
were infected and propagated the virus over the observation period.
Replication is analogous to the mouse fibroblast cell line L929. Compared
to RK-13 or ED cells that heavily propagate the virus but are lysed within
48 h (C), replication is low. All data are expressed as mean values³SD of
triplicates per group.
Dendritic cells (DC) are known to be the most potent
initiators of a T cell-dependent immune response (Steinman,
1991), and MHC class I-dependent antigen presentation has
generally been shown for DC, both for viral and tumour
antigens (Macatonia et al., 1988 ; Nair et al., 1992, 1993 ; Bender
DAAG
+ Virus, cell culture and virus assays. EHV-1 strain Ab4 (Gibson
et al., 1992 ; Telford et al., 1992) was propagated in RK-13 cells, grown in
Dulbecco’s modification of Eagle’s MEM (DMEM) with 5 % FCS.
Thereafter, virus was purified and accumulated by high-speed centrifugation (15 000 g, 15 min, Sorvall) and ultra-centrifugation through a
20 % sucrose layer (2 h, 100 000 g, Beckmann SW 27 rotor), respectively.
A virus pellet of 200 ml supernatent was resuspended in 4 ml NCM and
filtered sterile (0±45 µm, Sartorius). EHV-1 was titrated by plaque assay
using 24-well flat-bottom culture plates seeded with RK-13 cells. Virus
was inactivated by heating at 56 °C for 30 min, and then the suspension
was tested for surviving virus by plaque assay. The replication of EHV1 in DC in vitro was detected by infectious centre assays, co-culturing
twofold dilutions of DC with permissive RK-13 cells for 36 h before
counting the cytopathic lesions. To determine virus replication in the
lungs or brains of infected mice, total organs were collected and
homogenized in 1 ml tissue culture medium (DMEM and 5 % FCS). The
suspensions were serially diluted tenfold and virus titres were determined
by plaque titration on RK-13 cells. The results were expressed as the
geometric mean (p.f.u. per organ) obtained from six mice tested
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Fig. 2. Flow cytometric analysis of EHV-1-specific proteins and assessment of surface markers on DC upon EHV-1 infection.
(A) FSDC were infected with 1 p.f.u. per cell for 2 h and cultured for 5 days as indicated at the top of the peaks. An aliquot of
the cells was analysed daily for the surface expression of EHV-1-specific proteins. Faint expression of these glycoproteins can
already be detected within 24 h, thereafter the number of EHV-1-positive cells increases quite slowly as indicated besides the
x-axis. (B) FSDC were infected with 1–10 p.f.u. per cell, or pulsed with an equivalent amount of inactivated virus for 2 h,
cultured for 2 days and analysed for EHV-1-specific proteins. While pulsed cells failed to express EHV-1, the number of EHV-1positive cells increases with the infectious doses ; the slight difference in the fluorescence intensity, however, is not significant.
(C) Phenotypic analysis of EHV-1-infected DC. 80/1 DC were infected for 2 h with 100 p.f.u. per cell of live EHV-1 and cultured
for 24 h in their culture medium. Thereafter, untreated (control) or EHV-1-infected 80/1 DC were washed and stained with
appropriate MAbs. Filled profiles illustrate the reactivity of relevant MAbs, whereas open profiles represent irrelevant isotypematched control MAbs. Data shown represent one of at least three independent infections.
independently. Serum antibody titres were measured by indirect
immunofluoresence. Briefly, RK-13 cells in 96-well plates were infected
with 50 p.f.u. per well EHV-1 strain AB4. The cells were fixed with
formalin at the onset of plaque formation. Serum dilutions and an
appropriately diluted FITC-conjugated second-step reagent (goat antimouse FITC, Dianova) were incubated for 30 min each. Analysis was
performed with an Axiovert S 100 (Zeiss).
+ Infection of DC with EHV-1. To determine whether DC represent
a target for EHV-1, 10% DC were incubated for 2 h with 10% p.f.u. EHV1 strain Ab4. After two washings in NCM, the cells were cultured in 1 ml
of their specific culture medium for 5 days. A sample of the cells was taken
daily to determine the virus load by infectious centre assays. RK-13, ED
and L929 were infected as controls. The results are expressed as mean
³SD of triplicates. For the determination of surface markers and effects
on the T cell proliferation, 1–100 p.f.u. per cell were chosen for the 2 h
infection period.
+ Flow cytometry. To determine the expression of surface markers
on DC, FITC-conjugated anti-hamster MAbs 3E2 (anti-CD54) and 1610A1 (anti-CD80) were purchased from PharMingen. The anti-mouse
MAbs 11-4.1 (anti-H-2Kk) and 15-5-5S (anti-H-2Dk) were obtained from
Becton Dickinson and PharMingen, respectively. For the detection of
non-labelled MAb anti-H-2Kk and anti-H-2Dk, the FITC-conjugated
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F. Steinbach and others
second-step reagent goat F(ab«) anti-mouse IgG (H­L) (Caltag) was
#
used. In addition, the expression of viral proteins on the surface of
infected cells was examined using a polyclonal rabbit anti-EHV-1 serum
as described earlier (Slater et al., 1994 a). For the determination of surface
markers, DC were cultured 8–24 h prior to flow cytometric analysis. For
control purposes, cells were incubated with isotype-matched MAbs.
Stained cells were analysed using FACScan flow cytometers equipped
with LYSYS II and CellQuest software (Becton Dickinson).
+ T cell proliferation assay. 80}1 DC or control cells (L929) were
either infected with live EHV-1 (1–100 p.f.u. per cell) or pulsed with an
equivalent of heat-inactivated virus for 2 h at 37 °C in 1 ml NCM with
occasional agitation. After three washes, cells were X-irradiated
(1±5 Gy}min, Philips RT 305) with 40 Gy and seeded into 96-well roundbottom culture plates at 10% cells per well. Antigen presenting celldepleted, naive, syngeneic CD8+ T cells (97–99 % purity, as determined
by flow cytometry) were obtained according to a protocol described
elsewhere (Elbe et al., 1994) and 2¬10& T cells were added to each well
in either NCM or NCM supplemented with recombinant mouse IL-7
(6 ng}ml, Genzyme). At days 3, 4 and 5 of culture, cells were pulsed with
$H-TdR (37 kBq per well, Amersham) for 8 h and incorporation of the
radionucleotide was assessed by β-scintillation spectroscopy (Packard
Instruments). Data are expressed as mean values³SD of triplicates.
+ Vaccination and infection of mice. On days ®25, ®15 and
®5, DC (FSDC, D2SC1, 18, 80}1) or control cells (PAM212, J774, L929)
were pulsed with inactivated virus (5 p.f.u. per cell) for 90 min at 37 °C
in NCM with occasional agitation. Subsequently, each mouse was
immunized intranasally with 10& EHV-1-pulsed cells in a volume of 40 µl
medium. Control groups received NCM, the analogous amount of
inactivated virus or unpulsed DC only. On day 0, mice were challenged
intranasally with 10( p.f.u. live EHV-1. Clinical signs as well as the body
weight of all animals were monitored daily. To determine the amount of
virus replication in lungs or brains, two animals of each group were killed
on days 2 and 4, respectively. Experimental groups consisted of six mice
each.
+ Statistical analysis. StatView 4.5 was used to evaluate the
significance of experimental versus control group data.
Results
Fig. 3. DC either infected with EHV-1 or pulsed with heat-inactivated virus
stimulate naive, syngeneic CD8+ T cells in vitro. CD8+ T cells (H-2k ;
2¬105 per well) were co-cultured with syngeneic 80/1 DC (1¬104 per
well) which were infected for 2 h with (A) various doses (1–100 p.f.u. per
cell) of live EHV-1 strain Ab4, (B) 100 p.f.u. per cell of live EHV-1, or
pulsed for 2 h with the equivalent amount of heat-inactivated (hi) virus
(antigen-pulse). Results are representative of two experiments. In (C)
CD8+ T cells were cultured with syngeneic 80/1 DC or L929 that were
pulsed with heat-inactivated virus (100 p.f.u. per cell) in the presence or
absence of IL-7. Data shown are representative of four experiments. In all
experiments, the proliferative response was determined on day 4. Data are
expressed as mean values³SD of triplicates per group. T cells were
depleted of detectable accessory cells, as determined by their lack of a
ConA response (! 600 c.p.m.). Background counts with T cells in NCM
were ! 500 c.p.m., and ! 7500 c.p.m. in NCM supplemented with IL-7.
Background counts of irradiated 80/1 DC and L929 (infected, pulsed or
untreated and with/without IL-7) were ! 1500 and 3900, respectively.
Mouse DC can be infected with EHV-1
Our first set of experiments was designed to investigate
whether DC can be infected with EHV-1. As seen in Fig. 1 (A),
all DC lines propagated EHV-1 throughout the observation
period of 5 days. The replication rate per cell was rather low
(Fig. 1 B). Replication was analogous to the mouse fibroblast
cell line L929, but relatively low when compared to permissive
rabbit or horse cells (Fig. 1 C). Flow cytometric analysis of
EHV-1-infected FSDC revealed that some of the cells express
viral glycoproteins (Fig. 2 A, B). Faint expression of EHV-1specific proteins was already detected on 8–18 % of infected
FSDC after 24 h. The amount of proteins expressed as well as
the numbers of EHV-1-positive cells (ranging from 15–48 %)
increased only slowly thereafter (Fig. 2 A). Additionally, we
observed that the number of EHV-1-positive FSDC was
dependent on the amount of virus used for infection ; however,
minor differences with regard to the amount of protein
expressed were not significant. In contrast, FSDC pulsed with
DAAI
heat-inactivated virus failed to express EHV-1 proteins (Fig.
2 B).
EHV-1-pulsed 80/1 DC are potent stimulators of
naive, syngeneic CD8MT cells
The aim of the following experiments was to investigate
the role of DC in stimulating T cell immunity to EHV-1. We
have previously shown that the MHC class I+}II−}CD80+ DC
line 80}1 induces proliferation of naive, allogeneic CD8+ T
cells in an MHC class I-restricted fashion in vitro, but does not
stimulate proliferation of naive, allogeneic CD4+ T cells (Elbe
et al., 1994). The sensitizing power of 80}1 DC in vivo was
evidenced by the recent findings that these cells are able to
induce transplantation immunity and hapten-specific immune
responses (Lenz et al., 1996 ; Kolesaric et al., 1997). Thus, we felt
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Immunization with dendritic cells
Table 1. Clinical signs observed in mice and control animals after EHV-1 challenge
Groups are listed according to the genetic background of the mouse strains. Minor differences between
BALB}c and C3H mice occur in the markedness of the symptoms : while crowding and roughed fur are
stronger in BALB}c mice, ataxia was slightly more prominent in C3H mice. Roughed fur is also graded from
‘ ® ’ (no roughness) to ‘ ­­­ ’ (very strong roughness, affecting the total animal). Data shown summarizes
the observations from independent groups of six mice each. Roughed fur and crowding occurred from the
first day of infection. Ataxia occurred from day 4 on, so that only two mice in each set of experiments
remained (the others had served for determinations of virus titres in lung or brains). Animals died throughout
the observation period of 7 days. The number of dead animals includes those euthanized owing to terminal
illness. Control animals (BALB}c or C3H) were treated with medium, DC (FSDC or 80}1) or inactivated
EHV-1 (∆EHV-1) alone.
Mouse strain/
cell line
BALB}c medium
BALB}c FSDC
BALB}c ∆EHV-1
18
FSDC
D2SC1
J774
PAM212
C3H medium
C3H 80}1DC
C3H ∆EHV-1
80}1
L929
Onset of
clinical signs
(day no.)
Roughed
fur
Crowding
Ataxia
No. of dead
animals
1
1
1
2
2
1
1
1
1
1
1
2
1
­­­(12}12)
­­­(12}12)
­­­(18}18)
­ (6}18)
­ (7}18)
­­­(18}18)
­­­(18}18)
­­­(18}18)
­­ (12}12)
­­ (12}12)
­­ (18}18)
® (0}18)
­­ (18}18)
12}12
12}12
18}18
8}18
6}18
18}18
18}18
18}18
9}12
8}12
12}18
5}18
14}18
3}6
2}6
3}6
0}6
0}6
2}6
3}6
2}6
3}6
4}6
4}6
0}6
4}6
4}12
5}12
5}18
1}18
0}18
5}18
4}18
4}18
4}12
3}12
3}18
0}18
4}18
that this DC line could be an ideal tool to investigate whether
EHV-1-treated DC can stimulate naive, syngeneic CD8+ T
cells. We further asked whether an actual EHV-1 infection is
needed for an efficient presentation of viral antigens by these
cells and what amount of virus would provide the best results.
For this purpose, 80}1 DC were either infected or pulsed with
heat-inactivated virus, testing virus amounts from 1–100 p.f.u.
per DC. The infection neither changed their morphology nor
influenced the expression of MHC class I and the costimulatory molecules CD54 and CD80 (Fig. 2 C). Co-culturing
with highly purified naive, syngeneic CD8+ T cells revealed
that DC pulsed with heat-inactivated virus induced a stronger
proliferative T cell response than EHV-1-infected 80}1 DC,
with peak responses occurring with 100 p.f.u. per cell at day 4
of co-cultivation (Fig. 3 A, B). To determine whether other
MHC class I+}II− cells, when infected or pulsed with heatinactivated EHV-1, are also able to stimulate naive, syngeneic
CD8+ T cells in vitro, we tested the MHC class I+ fibrosarcoma
L929 in a similar experimental setting as described above. L929
cells infected or pulsed with heat-inactivated EHV-1 failed to
induce a significant proliferative response of naive, syngeneic
CD8+ T cells (Fig. 3 C and data not shown). This shows that
MHC class I expression on stimulator cells is not sufficient to
induce productive T cell responses and implies that the ability
of 80}1 DC to stimulate T cell proliferation in the absence of
MHC class II molecules is presumably due to the additional
delivery of co-stimulatory signals. Furthermore, the results
demonstrate that DC do not require actively replicating virus
to load their MHC class I molecules but, instead, inactivated
viral proteins brought into DC by non-infectious particles
suffice to activate CD8+ T cells. Based on a previous report
that IL-7 supports the activation and growth of in vitro antigenspecific CTL precursors (Ferrari et al., 1995), we added
recombinant IL-7 to the cultures of 80}1 DC, L929 cells and
CD8+ T cells. This led to a more vigorous T cell response to
EHV-1-pulsed, but not to unpulsed, 80}1 DC. In contrast,
IL-7 failed to augment the T cell response to EHV-1-pulsed
L929 cells (Fig. 3 C).
Protection against virus challenge following intranasal
immunization with EHV-1-pulsed DC
In order to test the in vivo activity of the DC lines, we used
two different mouse strains (BALB}c and C3H) corresponding
to the genetic background of the DC lines. Since the EHV-1driven T cell proliferation was superior with inactivated virus,
and the use of non-replicating EHV-1 together with DC might
offer advantages over live virus preparations (see Discussion),
the in vivo experiments were performed with DC pulsed with
heat-inactivated EHV-1. We used an immunization protocol
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F. Steinbach and others
Fig. 4. Mean weights of immunized animals on days 1 to 7 after challenge
with infectious EHV-1. Infected mice (A, BALB/c ; B, C3H) show two
different patterns of reaction. Weight curves of animals immunized with
EHV-1-pulsed control cell lines (PAM212, J774, L929) or DC line D2SC1
showed a steady decline in weight. Mice immunized with EHV-1-pulsed
DC lines FSDC, 18 and 80/1 showed only moderate loss in mean body
weight. Data are expressed as mean values ³SEM of six animals tested
independently. Co, heat-inactivated virus alone (control).
principally applicable to horses for potential candidates for
vaccinations and applied a relatively low amount of heatinactivated virus. Direct immunization of mice with inactivated
EHV-1 alone (equivalent of 5¬10& p.f.u. per animal and single
immunization) did not show any protective effects (controls in
Table 1 ; Figs 4 and 5) compared to medium-treated animals
(Table 1 ; Fig. 5). Thus, following EHV-1 challenge, these mice
became severely ill (crowding and loss of weight), some dying
from the infection (20–30 %) within the observation period of
8 days. In sharp contrast, animals immunized with DC lines
(FSDC, 18 or 80}1) that had been pulsed with heat-inactivated
virus, showed markedly reduced clinical symptoms (Table 1)
and significantly less weight loss (Fig. 4). Likewise, the virus
load in the lungs and CNS was reduced more than twofold (Fig.
5 A, B). From this panel of mice, only 1}54 died during the
observation period, implying that DC pulsed with inactivated
virus were able to protect mice against this potentially lethal
virus challenge, whereas DC alone (Table 1 ; Fig. 5) or the
virus-pulsed DC line D2SC1 did not exhibit any protective
effects. The latter finding is in keeping with results by other
investigators, showing that D2SC1 failed to induce CTL using
DABA
Fig. 5. EHV-1 replication in lungs and brains of BALB/c and C3H mice.
Data are shown with regard to the maximum replication of the virus on
days 2 (A, lungs) or 4 (B, brains). In both organs, infection was
significantly reduced by immunization with EHV-1-pulsed DC lines 18,
FSDC and 80/1, but not D2SC1. Bars represent geometric mean virus
titres per organ with SEM of six mice. Controls were performed with
medium alone, FSDC (BALB/c) or 80/1 (C3H) alone or heat-inactivated
virus alone. Asterisks indicate P ! 0±05.
Table 2. Appearance of serum antibodies after EHV-1
challenge
Mice were immunized with FSDC pulsed with heat-inactivated virus
or treated with medium only as described in Methods. After EHV-1
challenge, immunized mice developed serum antibodies more rapidly
and at a significantly higher titre. Data are expressed as mean of four
mice for each group.
Time (weeks)
Group
Control (medium)
FSDC
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1
2
4
! 1 : 10
! 1 : 10
! 1 : 10
1 : 40
1 : 10
1 : 80
Immunization with dendritic cells
soluble β-Gal and transfected cell lines as a model system for
tumour immunity, unless GM-CSF was provided additionally
(Paglia et al., 1996). Likewise, none of the control cells,
including the macrophage line J774 as an antigen presenting
cell effective in secondary responses (Bo$ hm et al., 1995),
showed any protective effects (Table 1 ; Fig. 4). Antibodies in
the serum appear only after the observation period of 1 week.
The MHC class II+ FSDC, however, support the induction of
such antibodies (Table 2), demonstrating that humoral immune
mechanisms were induced by the antigen-pulsed DC as well.
Discussion
DC are of growing interest for virus-directed immunological research. Considerable evidence exists that DC effectively present viral antigens to T cells, but their permissiveness
for EHV-1 was unknown. Using DC lines and the murine
model of EHV-1 infection, our results show that murine DC
may be sites for virus infections in addition to initiating an antiviral immune reaction.
EHV-1 infected all DC lines tested, but virus replication
was low. This correlates with data obtained by flow cytometry,
where only part of the cells expressed EHV-1-specific proteins
on their surface. Additionally, the infection appears to be nontoxic, since the DC are not lysed and remain viable for " 5
days. These data indicate that DC may mediate a kind of low
replicating persistent or restricted infection as previously
described for herpes simplex virus type 1 in monocytes}
macrophages (Linnavouri & Hovi, 1983 ; Domke-Opitz et al.,
1987 ; Morahan et al., 1989). This is in accordance with the
observation that the kinetics of replicating EHV-1 and
infectious titres obtained from the infected mouse cell line
L929 are much slower than in permissive rabbit or horse cells.
As 80}1 DC express MHC class I but no class II molecules
(Elbe et al., 1994), they provided a suitable tool to study the
initiation of a CD8+, MHC class I-restricted, EHV-1-specific
proliferation of T lymphocytes by DC. Surprisingly, stronger
proliferation of CD8+ T cells was obtained in vitro, when the
stimulating DC were pulsed with inactivated virus compared
to an actual EHV-1 infection of the DC. It remains to be
determined whether the inactivated EHV-1 gains access to the
classical, cytoplasmic machinery for processing and presentation on MHC class I molecules or whether the DC lines
used in this study employ alternative pathways for processing
exogenous antigens on MHC class I molecules (reviewed in
Jondal et al., 1996 ; Rock, 1996 ; Watts, 1997). Indeed, a nonclassical MHC class I processing pathway has been previously
described for heat-inactivated Sendai virus, which also
generated CTL responses in vivo (Liu et al., 1995). It is
conceivable that such a mechanism also takes place in our
model system. Thus far, the reason(s) for the relatively poor
EHV-1 presentation to T cells after infection is(are) not clear
because the expression of MHC class I (both H-2Kk and H2Dk) and co-stimulatory molecules (e.g. CD54, CD80)
remained unaffected post-infection. We presume that upon
infection with live EHV-1 the amount of MHC-associated
ligand is too small to elicit a strong CD8+ T cell-proliferative
response from naive T cells.
IL-7 has been described as an activation and growth factor,
being able to enhance the induction of CTL in vitro (Ferrari et
al., 1995 ; Welch et al., 1989 ; Kos & Mu$ llbacher, 1992, 1993)
and anti-viral CTL responses in vivo (Leong et al., 1997). Using
the MHC class I+}II− DC line 80}1 and naive CD8+ T cells,
the T cell stimulation was strongly enhanced when IL-7 was
provided to the cultures, confirming the described T cellactivating capacity of IL-7 in the context of antigen presentation by DC. Thus far, IL-7 has not been reported to be
synthesized by DC and it remains to be clarified whether it acts
on CD8+ cells alone or on DC as well. Since IL-7 induces the
secretion of IL-1α, IL-1β, IL-6 and TNF-α in monocytic cells
(Alderson et al., 1991) and IL-6 in turn acts as another growth
and differentiation factor for CTL (van Snick, 1990), the direct
action on DC may not be excluded. The potential involvement
of further cytokines in our system remains to be determined.
IL-12, for example, is known to stimulate CTL and co-operate
with IL-7 in the induction of human CTL (Mehrotra et al., 1993,
1995).
The successful intranasal administration of virus and
recombinant vaccines has been reported (Azmi & Field, 1993 a ;
Osterrieder et al., 1995) and seemed a suitable consideration for
a DC-based approach with EHV-1, especially since acute
infections had been demonstrated in lungs of mice and horses
(Awan et al., 1990 ; Gibson et al., 1992). The productive
infection of DC by EHV-1, however, could confer the problem
that infected DC, used for subsequent vaccination procedures,
would distribute the virus to mice, resulting in an indirect
vaccination with undefined amounts of live virus. Our results
indicate that it is possible to use DC lines pulsed with a low
amount of inactivated virus to elicit a significant immune
response in mice and protect them from a lethal challenge with
EHV-1. More than this, clinical signs were clearly reduced, as
seen from the body weights of the animals. Accordingly, the
virus titres in lungs and brains were reduced significantly, the
latter reduction arguing for the brain as a secondary target
organ. Anti-viral antibodies in the serum were only detectable
2 weeks after the infection and most likely did not contribute
to the initial protection mediated by the DC. This is in
accordance with earlier reports on intranasal EHV-1 vaccination (Azmi & Field, 1993 b ; Osterrieder et al., 1995 ; Bartels
et al., 1998), supporting our view that DC primarily elicited a
local immune response in this mouse model. Antibodies might,
however, contribute to a protection against reinfections.
A successful vaccination protocol for EHV-1 has been
described in mice, using high doses of live or subunit vaccines.
While live vaccines lead to a primary infection and might
establish latent or persistent infections, the use of virus
preparations or recombinant proteins requires amounts of
protein too large for a practical approach in horses (Tewari et
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DABB
F. Steinbach and others
al., 1994 ; Osterrieder et al., 1995). Accordingly, we have not
chosen the high amount of protein suggested by the in vitro
stimulation of naive CD8+ T cells but elected a low amount of
heat-inactivated virus in order to avoid any protective effect
by the virus preparation itself. Our results show that a strategy
using resident DC in vivo might be a much more powerful
vaccination tool. In this EHV-1 infection model, conflicting
data exist as to whether the immune response is CD4- or CD8mediated (Azmi & Field 1993 a ; Tewari et al., 1994). Although
the precise mechanism by which the DC-induced protection
occurs in vivo is unknown, it is likely that an initial CD8+ T cell
activity is responsible for the observed beneficial effects, since
at least DC lines 80}1 and 18 are MHC class II− and therefore
not able to activate CD4+ lymphocytes directly. An approach
to stimulate one of these T cell subsets with freshly isolated
MHC class I+, class II+ DC, while depleting the other by MAbs
in vivo might help to elucidate the mechanisms taking part in
the induction of this protection.
Although definitive experimental proof is lacking, we
propose that intranasally applied DC, aspirated into the
tracheo-bronchial system, can migrate via afferent lymphatics
to the lymph nodes in numbers sufficient to initiate an antiviral response. Evidence obtained in preliminary migration
studies (F. Steinbach, unpublished data) supports this assumption. This concept fits the biology of airway-DC and earlier
investigations on the interaction of airway-DC with pathogens
(Schon-Hegrad et al., 1991 ; Holt et al., 1994 ; McWilliam et al.,
1996 ; Semper & Hartley, 1996). In summary, we have shown
that DC pulsed with heat-inactivated EHV-1 virus can
successfully be used for in vivo vaccination in mice. The in vitro
data using IL-7 raise the possibility that the in vivo responses
may even be improved by the addition of IL-7 or yet
undetermined cytokines supplied as adjuvants or delivered as
gene therapy products.
We thank Dr W. Phares for critical reading of the manuscript and T.
Leiskau and S. Olt for excellent technical assistance. This work was
supported by grants from the Austrian Science Foundation [P10797MED (AEB)], the German Science Foundation (Bo 1005}1-3) and from
the State of Berlin (FSt). F. S. and A. E.-B. contributed equally to this work.
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