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J Virol, June 1998, p. 4866-4873, Vol. 72, No. 6
Federal Research Centre for Virus Diseases of
Animals1 and
Institut für
organische Chemie,
Received 29 October 1997/Accepted 17 February 1998
Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy
economic losses in the pig industry. Therefore, vaccination with live
attenuated viruses is practiced in many countries. This vaccination
was demonstrated to induce extrathymic virus-specific memory
CD4+CD8+ T lymphocytes. Due to their major
histocompatibility complex (MHC) class II-restricted proliferation, it
is generally believed that these T lymphocytes function as memory
T-helper cells. To directly prove this hypothesis, 15-amino-acid,
overlapping peptides of the viral glycoprotein gC were used for
screening in proliferation assays with peripheral blood mononuclear
cells of vaccinated d/d haplotype inbred pigs. In these
experiments, two naturally processed T-cell epitopes (T1 and T2) which
are MHC class II restricted were identified. It was shown that
extrathymic CD4+CD8+ T cells are the
T-lymphocyte subpopulation that responds to epitope T2. In addition, we
were able to show that cytokine secretion can be induced in
these T cells through recall with inactivated PRV and demonstrated
that activated PRV-primed CD4+CD8+ T cells
are able to induce PRV-specific immunoglobulin synthesis by
PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the
priming of humoral anti-PRV memory responses. The
experiments identified the first T-cell epitopes so far known
to induce the generation of virus-specific
CD4+CD8+ memory T lymphocytes and showed that
CD4+CD8+ T cells are memory T-helper
cells. Therefore, this study describes the generation of virus-specific
CD4+CD8+ T cells, which is observed during
vaccination, as a part of the potent humoral anti-PRV memory response
induced by the vaccine.
Pseudorabies virus (PRV), a member
of the Alphaherpesvirinae, is the causative agent of
Aujeszky's disease. This disease is lethal to young pigs and causes
important economic losses (52). Therefore, vaccination of
pigs is practiced in many countries.
Several humoral immune system effector mechanisms are involved in the
protection of pigs from PRV infection. Virus-neutralizing antibodies,
antibodies mediating antibody-dependent cell-mediated cytotoxicity, and
antibodies mediating complement-mediated lysis of PRV-infected target
cells have been demonstrated (22, 23, 53, 54). The main
targets of this humoral immune response were shown to be the
viral glycoproteins (3, 45), and passive immunization
with monoclonal antibodies (MAbs) against gB, gC, and gD protects
pigs from a lethal challenge (20, 49).
The protection conferred through cell-mediated immunity is
poorly understood. An increase in major histocompatibility
complex (MHC)-unrestricted cell-mediated cytotoxicity against
uninfected and PRV-infected cells has been detected after
infection or vaccination of pigs with PRV (16, 53, 54), and
specific cellular immune responses to PRV infections could be
demonstrated by stimulation of proliferation and lymphokine secretion
of porcine PRV-immune lymphocytes (10, 17, 42, 43, 51) as
well as by the detection of PRV-specific cytotoxic lymphocytes
(21, 56).
There are some difficulties in defining more precisely the impact of
cell-mediated immune effector mechanisms to protection from
PRV-infection and their interplay with the observed humoral immune
response. Considerably fewer porcine than human or mouse differentiation markers are available (34). In addition, the immune system of swine differs considerably from that of humans and
mice. The pig has a substantial number of
CD4 To gain a better understanding of immune effector mechanisms conferring
protection from PRV infection, the function of these unusual
extrathymic T-lymphocyte subsets has to be elucidated. In the present
study, we identified two T-cell epitopes on glycoprotein gC which
are primed during vaccination of d/d haplotype inbred pigs
(41) against PRV and demonstrated that MHC class
II-restricted, peripheral CD4+CD8+ memory
T lymphocytes are the responding T lymphocytes. We were further
able to show that PRV-specific, extrathymic
CD4+CD8+ T lymphocytes are able to secrete
cytokines and have the capacity to stimulate the secretion of
PRV-specific immunoglobulins (Ig) by PRV-primed B cells. These results
demonstrate that porcine CD4+CD8+ T lymphocytes
can function as memory T-helper cells and can direct humoral anti-PRV
memory responses.
Animals, immunizations, and challenge.
Three, 4- to
24-month-old d/d haplotype NIH miniature pigs
(41) were immunized twice at an interval of 10 days with the PRV vaccine Nobi-Porvac live (Intervet, Boxmeer, The Netherlands) as
recommended by the manufacturer. Blood samples were collected before
and 2 to 24 months after immunization.
MAbs.
Murine MAbs against porcine CD4 (MAb 74-12-4 [27]), MHC class I (MAb 2.27.3a [13,
26]), and MHC class II DR (MAb MSA3 [11, 19])
were kindly provided by J. K. Lunney (USDA Agricultural Research
Service, Beltsville, Md.). A murine MAb against porcine IgM (MAb 2E8
[2]) was kindly provided by M. Amadori (Instituto Zooprofilattico Sperimentale, Brescia, Italy). Murine MAbs against porcine IgG, IgG1, and IgG2 (47) were a generous gift of
A. T. Bianchi (Central Veterinary Institute, Lelystad, The
Netherlands). The murine MAbs against porcine CD8 (MAb 295/33
[14]) and SWC1 (MAb 8/1 [33, 37])
were established at the Bundesforschungsanstalt in Tübingen.
Cell lines and cell culture.
The porcine kidney cell line
PSEK (American Type Culture Collection, Rockville, Md.) and peripheral
blood mononuclear cells (PBMC), isolated from blood with a Ficoll
gradient, were cultivated in RPMI 1640 medium supplemented with 10%
(vol/vol) fetal calf serum, 10 mM HEPES [pH 7.0], 2 mM
L-glutamine, 100 U of penicillin per ml, 0.1 mg of
streptomycin per ml, and 5 × 10 Virus preparation.
The PRV strain Phylaxia was propagated on
PSEK cells. The viral stocks were clarified and either subjected to
titer determination by plaque assay and subsequently UV inactivated as
described previously (43) or sedimented at 70,000 × g and resuspended in phosphate-buffered saline to prepare
partially purified virions. The protein content of the virion
suspension was subsequently determined with a protein quantitation kit
(Bio-Rad, Munich, Germany) and a bovine serum albumin standard.
Synthesis and analysis of synthetic peptides.
Overlapping
peptide amides of glycoprotein gC (31), 15 amino acids in
length and overlapping by 10 amino acids, were synthesized by Fmoc
(9-fluorenylmethoxycarbonyl) chemistry (multiple peptide synthesizer
SMPS 350 A.; Zinsser, Frankfurt, Germany). The synthesized peptides
were analyzed by amino acid analysis (ABI 420A; Applied Biosystems,
Weiterstadt, Germany), analytical high-pressure liquid chromatography
(System Gold, Beckman, San Ramon, Calif.) on a Nucleosil
C18 column (Grom, Herrenberg, Germany), and ion-spray mass
spectrometry (API III Triple-Quatrupol ion spray MS [Grom]).
PRV ELISA.
Partially purified PRV in phosphate-buffered
saline was spread onto 96-well microtiter plates at a protein
concentration of 5 µg/well, and a standard enzyme-linked
immunosorbent assay (ELISA) was performed as described previously
(21). Bound PRV-specific antibodies were either detected
with rabbit anti-pig Ig heavy plus light chains (H+L) conjugated to
horseradish peroxidase (HRP) or with isotype-specific murine MAbs
against IgM, IgG, IgG1, and IgG2 followed by goat anti-mouse Ig
(H+L)-HRP, which was preincubated with an equal volume of preimmune
swine serum to eliminate cross-reactivity. The ELISA titer was
determined as the highest dilution of the serum displaying more than
0.1 difference in optical density at 490 nm from the corresponding
preimmune serum.
Virus neutralization assay.
The virus-neutralizing activity
of the antisera was tested in a 50% plaque reduction assay
(26) performed on MDBK cells in the absence or presence of
5% rabbit serum as a source of complement. The neutralization titer
was expressed as the dilution of serum giving 50% plaque reduction.
Stimulation of PBMC.
To screen for T-cell epitopes with
overlapping peptides, 105 PBMC were stimulated with
different concentrations of peptide (ranging from 0.6 to 0.0005 mg/ml)
for 4 days. Proliferation was subsequently measured by adding
[3H]thymidine (1 µCi/well) for 18 h to the
cultures followed by harvesting onto fiberglass filters, which were
then analyzed in a scintillation counter as previously described
(38). Virus-specific stimulation of PBMC was analyzed by
adding UV-inactivated virus to the cell culture at multiplicity of
infection of 10 (determined before inactivation). For stimulation of T
lymphocytes or subpopulations of T lymphocytes, 30-Gy-irradiated
autologous PBMC were added (105/well) to ensure antigen
presentation. All experiments were performed in triplicate. For a
better comparison between different experiments, the stimulation
coefficient (ks) was calculated as follows:
ks = (mean of peptide specific stimulation/mean
of spontaneous proliferation) Two-color flow cytometric analysis and cell sorting.
Two-color staining of cells for CD4 and CD8 was performed as described
previously (43). For fluorescence-activated cell sorting of
T lymphocytes, monocytes were depleted by plastic adherence and B
lymphocytes were removed by passages over nylon wool columns (39). Cell separation was performed by setting electronic
sort windows on the four CD4/CD8-defined T-lymphocyte subpopulations. The purity of the separated fractions was always higher than 98%.
Measurement of T-cell-dependent Ig synthesis of B
lymphocytes.
PBMC of a PRV-vaccinated d/d haplotype
inbred pig were stimulated for 4 days with UV-inactivated PRV-virus at
an MOI of 2 and subsequently enriched for T lymphocytes on nylon wool.
The activated T lymphocytes and B lymphocytes of a PRV-immunized
d/d haplotype inbred pig, negatively selected by
immunomagnetic cell separation with anti-SWC1, were cocultivated for 7 days at different T-cell/B-cell ratios (25 to 1.5 × 104 T cells/ml and 1 × 106 B cells/ml) in
a final volume of 200 µl. Finally, the supernatants were tested for
anti-PRV Ig production in a PRV ELISA.
Measurement of cytokine secretion.
Nylon wool-purified T
lymphocytes (105 cells), together with 30-Gy- A T-cell-dependent humoral immune response is induced by the live
vaccine.
Three d/d haplotype inbred pigs were immunized
twice with the live PRV vaccine Nobi-Porvac live. To monitor the
humoral immune response, blood samples were collected before and at
different times (starting at 2 months) after immunization and assayed
in ELISA for anti-PRV reactivity. Figure
1 shows the representative analysis of
the observed secondary humoral anti-PRV response. PRV-specific
antibodies in immune but not in preimmune sera and antibodies to bovine
serum albumin were detected up to a serum dilution of at least 1:10,000
(Fig. 1a). The induction of anti-PRV antibodies by the vaccine could
also be confirmed by virus neutralization assays in the presence and
absence of complement, which exhibited serum neutralization titers of
1:500 and 1:2,000, respectively (data not shown).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Vaccine-Induced, Pseudorabies Virus-Specific,
Extrathymic CD4+CD8+ Memory T-Helper Cells
in Swine
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
CD8 T lymphocytes in the peripheral
blood (4, 6, 12, 36, 39). In young animals, this
subpopulation of T lymphocytes comprises up to 60% of the T
lymphocytes and contains mainly 
T lymphocytes. The pig is also
the only species so far known to contain a substantial number of
resting extrathymic CD4+CD8+ T lymphocytes
(28, 36, 39). This T-lymphocyte population shows
morphologically the phenotype of mature T lymphocytes (40) and increases with age to up to 60% of peripheral T lymphocytes (29, 35, 39, 55). Further, it was demonstrated that
CD4+CD8+ T lymphocytes comprise memory T cells
which proliferate upon stimulation with recall antigen (43,
55). Since the observed proliferative response was shown to be
MHC class II-restricted, it was speculated that the porcine
CD4+CD8+ T-cell subset contains memory T-helper
lymphocytes (43). However, the ability of these T
lymphocytes to secrete cytokines or to provide help to B cells has so
far not been demonstrated.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
5 M
2-mercaptoethanol. The medium for the murine interleukin-2 (IL-2)-dependent cell line HT-2 was additionally supplemented with 10 IU of recombinant human IL-2 (Boehringer, Mannheim, Germany) per ml.
The bovine kidney cell line MDBK (American Type Culture Collection),
used for virus titer determination, was cultivated as described
previously (18). All cells were incubated at 37°C in a
humidified atmosphere containing 5% CO2.
1.
-irradiated
autologous PBMC (105/well) to ensure antigen presentation
were restimulated for 3 days, and the cell culture supernatant was
collected. The supernatant (100 µl) was subsequently added to 5 × 103 HT-2 cells in a final volume of 150 µl of medium
without IL-2 and incubated for 24 h. After addition of
[3H]thymidine (1 µCi/well) and an 18-h incubation, the
cells were harvested onto fiberglass filters, which were then analyzed
in a scintillation counter as previously described (38).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (31K):
[in a new window]
FIG. 1.
The PRV live vaccine induces anti-PRV antibodies of the
IgG isotype. (a) Partially purified PRV (5 µg/well [circles and
squares]) or bovine serum albumin (triangles) was coated and the
reactivity of a PRV-immune (solid) and a preimmune (open) serum of a
d/d haplotype inbred pig after the second vaccination was
determined by an ELISA. (b to d) Detection of anti-PRV Igs of the IgM
(b), IgG (c), and IgG1 and IgG2 (d) isotypes in an ELISA. Anti-PRV
reactivity (5 µg of partially purified PRV per well) of PRV-immune
(stippled bars) and preimmune (open bars) sera at a dilution of 1:2,000
(d) or 1:5,000 (b and c) detected by MAbs against the respective
isotypes or an appropriate control antibody (control) is shown. The
standard deviation of the single experiments is indicated by error
bars. OD490, optical density at 490 nm.
Identification of the PRV-specific T-cell epitopes T1 and T2. To identify T-cell epitopes which prime anti-PRV immune responses during vaccination, we used proliferation assays with PBMC of PRV-vaccinated d/d haplotype inbred pigs. With virus-derived, Escherichia coli-expressed fusion proteins for stimulation, preliminary results indicated the presence of epitopes on the viral glycoprotein gC (data not shown) which are naturally processed during vaccination. To delineate these epitopes, 94 overlapping peptides spanning the whole gC region were synthesized and tested for their ability to stimulate proliferation.
No specific proliferation could be detected for peptides covering amino acids 1 to 230 (peptides 1 to 46) and 420 to 479 (peptides 82 to 94) (data not shown). However, as shown for a representative experiment in Fig. 2, stimulation of PRV-primed PBMC with peptides 245, 355, and 360 identified two immunodominant T-cell epitopes (T1 and T2) between amino acids 230 and 420 of gC. All three peptides reproducibly showed stimulation coefficients (ks) of ca. 2, significantly higher than background (ks = 0 to 0.5). The observed stimulation was concentration dependent and reached its maximum (ks = ca. 3) at a peptide concentration of ca. 50 µg/ml (data not shown). PRV-primed PBMC did not proliferate in response to irrelevant peptides, and the identified PRV-specific peptides did not stimulate autologous nonimmune PBMC (data not shown). This demonstrated that the identified T-cell epitopes stimulate proliferation in a PRV-specific manner. Additional experiments showed, further, that both epitopes stimulated the proliferation of PBMC of all three vaccinated inbred pigs. Epitope T2 thereby elicited a stronger response than did epitope T1 at optimal peptide concentrations (data not shown).
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Epitope T2 stimulates PRV-specific MHC class II-restricted CD4+CD8+ memory T lymphocytes. To obtain more information about the function of T lymphocytes which are primed by the identified T-cell epitope T2 during vaccination, we determined the MHC restriction of the proliferative response to peptide 355. After adding MAbs against CD4, CD8, MHC class I, and MHC class II molecules, we tested the inhibition of peptide-specific proliferation of PRV-primed T lymphocytes by the MAbs. As shown in Fig. 3b, only antibodies against the CD4 coreceptor and MHC class II molecules inhibited proliferation induced by the peptide by more than 80% in comparison to cultures without antibodies added. Antibodies to CD8 and MHC class I molecules, for which their blocking capacity was demonstrated previously (14, 24, 26), did not show a significant inhibition. Therefore, it could be concluded that presentation of the epitope T2 is MHC class II restricted and that only the CD4 coreceptor is essential for the recognition of the peptide. We further compared the inhibition of peptide-specific proliferation through MAbs against CD4 and MHC class II with the inhibition of PRV-specific proliferation observed with the same antibodies. As shown in Fig. 3a, both antibodies also inhibited stimulation with UV-inactivated PRV by more than 80% (Fig. 3a). This indicated that peptide 355 might stimulate the same T-lymphocyte population as UV-inactivated PRV does.
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CD8+,
CD4+CD8, CD4CD8,
and CD4+CD8+ (Fig.
4a). The proliferative response of the
different T-lymphocyte subsets to recall with peptide 355 was
subsequently determined and compared to proliferation obtained upon
stimulation with UV-inactivated PRV (Fig. 4b). To exclude a toxic
effect of the MAbs used for separation, we labeled the whole
T-lymphocyte fraction with anti-CD4 and anti-CD8 prior to antigenic
stimulation and demonstrated that this did not influence the ability of
the T lymphocytes to proliferate in response to the different stimuli
(Fig. 4c). Subsequently, we demonstrated for the single T-lymphocyte
subsets that neither CD4CD8 (data
not shown) nor CD4CD8+ (Fig. 4b, panel I) T
lymphocytes showed peptide-specific proliferation. Also, T lymphocytes
with the phenotype of classical T-helper cells (CD4+CD8) did not show a statistically
significant increase in proliferation (Fig. 4b, panel III). A specific
proliferative response to peptide 355 (T2) was detected only for the
CD4+CD8+ T-lymphocyte subpopulation (Fig.
4b, panel II). In addition, only the CD4+CD8+
T-lymphocyte population showed a distinct enrichment of the responding cells in comparison to the nonseparated MAb-labeled T lymphocytes before sorting (Fig. 4c, MAb-labeled). The comparison of the
peptide-induced proliferative response with the response induced by
UV-inactivated PRV (Fig. 4b and c) demonstrated further that
peptide 355 and inactivated PRV stimulated the same subset of T
lymphocytes. Only CD4+CD8+ T lymphocytes
showed a significant proliferative response, as well as an enrichment
of responding cells upon sorting, with both stimuli (Fig. 4b, panel
II). Since PRV-specific CD4+CD8+ T
lymphocytes have been shown to be memory T lymphocytes (43, 55), these results demonstrate that epitope T2 primes
CD4+CD8+ memory T lymphocytes during
vaccination.
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PRV-specific memory T-lymphocytes stimulate the secretion of cytokines and can provide help for Ig-secretion by B cells. The restriction of the proliferative response against epitope T2 and UV-inactivated PRV (Fig. 3) indicated that PRV-specific CD4+CD8+ T cells are memory T-helper lymphocytes. One of the functions of T-helper lymphocytes is the secretion of cytokines (1, 44). Therefore, the supernatants of PRV-primed T lymphocytes after stimulation with inactivated PRV (only CD4+CD8+ T lymphocytes respond to this stimulus [Fig. 4b]) were tested in a bioassay with the murine IL-2-dependent cell line HT-2. As shown in Fig. 5a, HT-2 cells proliferated only in response to supernatants derived from PRV-primed T lymphocytes stimulated with inactivated PRV but not to supernatants of unstimulated T lymphocytes. In addition, no PRV-specific proliferation in response to supernatants derived from autologous nonimmune T lymphocytes stimulated with inactivated PRV was seen (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Much progress has been made during the last few years in
elucidating different immune mechanisms primed during PRV infection in
pigs as well as in generating new anti-PRV vaccines. However, although
currently available vaccines are able to protect pigs from a
lethal infection, they still allow the establishment of a latent
infection and the secretion of reactivated virus (32). The
development of more efficient vaccines is hampered by a lack of precise
knowledge of the different immune mechanisms which can confer
protection. In this study, three d/d haplotype inbred pigs
(41) were vaccinated with the commonly used PRV live vaccine Nobi-Porvac live (Tk/gE
[48]) and used to study vaccine-induced
protection against PRV on a defined genetic background.
The analysis of the humoral immune response in d/d haplotype inbred pigs demonstrated the presence of anti-PRV-specific antibodies with complement-dependent and independent virus-neutralizing activity. As demonstrated in outbred pig population with different live and killed PRV vaccines (15), in inbred pigs the main serum isotype of anti-PRV antibodies was IgG, and PRV-specific antibodies of the IgG1 and IgG2 isotypes could be detected. Thus, PRV vaccines generally seem to induce a long-term humoral memory response against PRV, which is characterized by efficient isotype switching to IgG. The observed isotype switch also indicated that a T-cell-dependent memory response was generated during vaccination (8).
In humans and mice, there are two functionally distinct
classes of extrathymic memory T lymphocytes with the ability to
proliferate upon contact with protein antigens. (i)
CD4CD8+ cytotoxic T lymphocytes are MHC
class II restricted and mainly recognize replicating viral antigens.
(ii) CD4+CD8 helper T-lymphocytes are MHC
class II restricted and respond to nonreplicating protein antigens
which have been processed by antigen-presenting cells (9, 46,
50). Conclusively, it was found that in pigs the proliferative
response of PRV-primed T lymphocytes upon recall with UV-inactivated
virus is MHC class II restricted and requires the CD4 coreceptor for
recognition (28, 43).
So far, several studies implicated an involvement of glycoprotein gC in the induction of PRV-specific MHC class I- and class II-restricted memory responses. First, it was shown that vaccination of pigs with a recombinant gC subunit vaccine or gC vaccinia virus recombinants leads to a significant protection of pigs from PRV-infection (30, 31a). Second, cells expressing PRV gC could be used for in vitro restimulation of lymphocytes from PRV-immune inbred pigs of the same haplotype (17). Third, it was shown that PRV infection induces a significant amount of MHC class I-restricted and gC-specific cytotoxic activity by T cells against PRV-infected target cells in two Landrace pigs (56). In this study, we focused on the precise characterization of the MHC class II-restricted memory T-lymphocyte response and virus-specific components which are able to prime this response. Since it is well known that MHC class I and class II molecules present peptides of 8 to 9 and 11 to 30 amino acids, respectively, in humans and mice (46), we selected for MHC class II-restricted T-cell epitopes by using peptide screening with overlapping 15-amino-acid peptides of viral glycoprotein gC. This approach allowed us to map two T-cell epitopes (T1 and T2) on gC, which could subsequently be shown to be MHC class II restricted and to require the CD4 coreceptor for recognition.
There were several lines of evidence that resting peripheral
CD4+CD8+ T lymphocytes play an important
role in protection against PRV-infection. The CD4/CD8 depletion studies
of Kimman et al. (17) indicated that
CD4+CD8dull+ T lymphocytes might contribute
to the lymphoproliferative response to UV-inactivated virus. Moreover,
Summerfield et al. (43) and Zuckermann and Husmann
(55) demonstrated through in vitro restimulation of the four
different extrathymic T-lymphocyte subsets with UV-inactivated PRV that
MHC class II-restricted memory T lymphocytes of the
CD4+CD8+ phenotype are generated during PRV
infection. These memory T lymphocytes were further shown to require
only the CD4 (not the CD8) coreceptor for recognition (43).
Since the restriction of the identified epitopes was in agreement
with proliferating CD4+CD8 as well as
with CD4+CD8+ T lymphocytes, we determined
the responding T-lymphocyte subset to T2. We could demonstrate that
only resting CD4+CD8+ T lymphocytes
proliferated significantly in response to this epitope. Since
PRV-specific CD4+CD8+ T lymphocytes were
shown to be memory T lymphocytes (43, 55), these results not
only identified the first T-cell epitope so far known to prime this
unusual extrathymic T-lymphocyte subset but also indicated that a PRV
live vaccine induces a long-lived MHC class II-restricted memory
T-lymphocyte response to the glycoprotein gC. This
interpretation was further supported by the observation that
stimulation of proliferation through epitope T2 was seen with blood
samples up to 12 months after the second administration of the vaccine.
We also compared the stimulation with the gC-derived peptide (T2) to
the stimulation with inactivated PRV and demonstrated that both stimuli
have the same restriction and activate the
CD4+CD8+ T lymphocytes exclusively. This is
in contrast to results described by Zuckermann and Husmann
(55), where in addition to double-positive memory T
lymphocytes a significant number of
CD4+CD8 memory T-lymphocytes proliferated
in response to UV-inactivated PRV. However, in several experiments we
did not detect a statistically significant proliferation of the
CD4+CD8 T-lymphocyte subset upon
stimulation with gC-derived peptide or inactivated virus, nor did we
observe an increase of proliferation upon cell sorting. Differences in
the inbred haplotypes used for the experiments (c/c versus
d/d haplotype animals), in the immunization protocol, and in
the time points when blood samples were taken during the two studies
might account for this discrepancy. In our study, we worked with
d/d haplotype swine and used blood samples collected 2 to 12 months after the second immunization. These conditions should have
favored the selective detection of a long-lasting memory response.
Although it could be shown that the CD4+CD8+ T-lymphocyte subset contains memory T lymphocytes (43, 55) and that the proliferation of these memory T cells is MHC class II restricted and can be inhibited by antibodies against CD4 (43), a direct demonstration of their helper activity was missing. A precondition of memory T-helper lymphocyte function is the ability to secrete cytokines and to provide help to B cells (1). Since only CD4+CD8+ memory T lymphocytes proliferated in response to stimulation with UV-inactivated virus in our system, by using PRV-primed T lymphocytes, we were able to demonstrate the virus-inducible production of cytokines.
Additionally, we established an assay for the detection of PRV-specific
Ig-synthesis by resting PRV-primed B cells upon stimulation with
activated, autologous, PRV-primed T cells. This assay allowed us to
directly link the vaccine-induced generation of PRV-specific CD4+CD8+ memory T lymphocytes and the
potent humoral anti-PRV memory response, which we detected in
vaccinated d/d haplotype inbred pigs. Our demonstration of
antibody synthesis in this assay, together with the MHC class II
restriction of the proliferative response and the ability of PRV-primed
CD4+CD8+ T cells to secrete cytokines,
identified these T lymphocytes as memory T-helper cells. It indicates
that they regulate the humoral memory response to PRV in vivo. The
resting phenotype (39) of
CD4+CD8+ T lymphocytes, the undetectable
expression of IL-2 receptor on the cell surface (40a), as
well as the high 
1-integrin level of 75% (55) can
therefore be interpreted as a prerequisite to fulfill their memory
T-helper functions. However, in contrast to murine and human memory
T-helper lymphocytes, resting porcine extrathymic
CD4+CD8+ T lymphocytes also express
significant levels of MHC class II molecules (40) and
can act as antigen-presenting cells in mixed leukocyte
culture (38). This indicates that the characterized resting, PRV-primed CD4+CD8+ T
lymphocytes may not only act as memory T-helper cells but may also be
able to act as antigen-presenting cells and may therefore display a
higher regulatory potential than the
CD4+CD8 memory T-helper lymphocytes
described in humans and mice.
Taken together, this study demonstrated that vaccination against PRV induced a potent T-cell-dependent humoral memory response. It mapped two naturally processed T-cell epitopes to glycoprotein gC, which are able to prime MHC class II-restricted memory T lymphocytes of the CD4+CD8+ phenotype. Our demonstration that virus-specific CD4+CD8+ cells can function as memory T-helper cells allowed us to identify the generation of PRV-specific CD4+CD8+ memory T lymphocytes during vaccination as being part of the potent humoral immune response, which is induced by the vaccine.
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ACKNOWLEDGMENTS |
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We thank S. Maurer for technical assistance and B. Teufel for help with peptide synthesis. We also thank W. Beck, M. Munari, and G.-J. Nicholson for acquiring the spectra for the peptide analysis.
This work was partially funded by grant DFG-Rz 2/1-5 from the Deutsche Forschungsgemeinschaft and by grant BRIDGE BIOT-CT91 from the European Community.
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FOOTNOTES |
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* Corresponding author. Mailing address: Bundesforschungsanstalt für Viruskrankheiten der Tiere, Paul-Ehrlich Strasse 28, 72076 Tübingen, Germany. Phone: 49-7071-967256. Fax: 49-7071-967303. E-mail: armin.saalmueller{at}tue.bfav.de.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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