From the Institut für Immunologie,
Bundesforschungsanstalt für Viruskrankheiten der Tiere and
¶ Interfakultäres Institut für Zellbiologie,
Abteilung Immunologie, 72076 Tübingen, Germany
Received for publication, October 30, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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The first naturally processed peptide synthesized
by a virus and recognized by classical CD8+ T cells
in association with the RT1.Al major histocompatibility
complex class I molecule of the Lewis rat is reported. Borna
disease virus-specific CD8+ T cells recognize
syngeneic target cells pulsed with peptides extracted from Borna
disease virus-infected cells. The predicted peptide sequence ASYAQMTTY
from the viral p40 protein coeluted with the cytotoxic
T-lymphocyte-reactive fraction was identified among natural ligands by
tandem mass spectrometry. Numerous naturally processed peptides derived
from intracellular bacteria, viruses, or tumors and recognized by
CD8+ T cells of man and mice are known, leading to a better
understanding of cellular immune mechanisms against pathogens in these
two species. In contrast, for the rat little information exists with
regard to the function and role of CD8+ T cells as part of
their cellular immune defense system. This first naturally processed
viral epitope in the rat contributes to the understanding of the rat
cellular immune response and might trigger the identification of more
cytotoxic T-lymphocyte epitopes in this animal.
Major histocompatibility complex (MHC)1 class I
molecules present peptides to CD8+ T cells, which recognize
this complex by their T cell receptor. Such CD8+ cytotoxic
T-lymphocytes (CTLs) detect cells
infected with viruses or intracellular bacteria and consequently
destroy these infected cells by cytotoxic effector mechanisms (1-4).
MHC class I molecules are assembled by combining the The rat expresses two different types of MHC class I molecules, a
classical class Ia, which is responsible for conventional antigen
presentation to CTLs, and nonclassical class Ib molecules with
unconventional or undefined functions (9, 10). The RT1.A region of the
rat MHC encodes for the class Ia molecules, whereas different RT1
regions encode the nonclassical class Ib molecules (11, 12).
Furthermore, in rats, unlike humans or mice, two functionally allelic
forms of the TAP exist, which are called TAP-A and TAP-B. These
molecules can be distinguished by their different peptide transport
specificities. In the Lewis rat, RT1.Al molecules are
linked to TAP-A (reviewed in Ref. 13). To our knowledge, the motifs of
only two TAP-A-associated molecules, RT1.Al and
RT1.Aa, have been determined (14-16), allowing epitope
prediction for these class I molecules. Thus far, only peptides from
naturally processed self-proteins are known to bind to
RT1.Al and RT1.Aa molecules, whereas no
information on peptides associated with class I molecules from
intracellular bacteria or viruses exists.
One of the few experimental infectious models in rats is Borna disease,
caused by Borna disease virus (BDV), a noncytolytic single-stranded RNA
virus, belonging to the order of Mononegavirales. BDV-induced
Borna disease is an encephalomyelitis originally described in horses
and sheep (17, 18). In recent years, this viral infection of the
central nervous system has been diagnosed in a wide variety of animals
including cattle, cats, dogs, and birds (reviewed in Ref. 19).
Furthermore, Borna disease virus, its nucleic acid, and specific
antibodies were detected in the blood of patients with psychiatric
diseases (20-25). However, there is no evidence whether BDV represents
the causative agent for any human disorder.
The best investigated animal model for the pathogenesis of BDV
infection is the Lewis rat. After intracerebral infection, the animals
develop an encephalomyelitis in which the infiltrating cells have been
characterized as CD4+ and CD8+ T cells and
macrophages (26, 27). BDV-specific CD8+ T cells represent
the effector cell population during the acute phase of the disease and
significantly contribute to the destruction of virus-infected brain
cells in vivo. Moreover, evidence has been presented that
this T cell population also participates in the degenerative
encephalopathy resulting in a severe cortical brain atrophy in the
chronic phase of the disease (28-30). Besides their role in
immunopathology, however, BDV-specific CD8+ T cells were
also known to eliminate the virus, without causing disease.
BDV-specific CD4+ T cells given prior to infection induce
CD8+ T cells, which eliminate the virus without causing
significant cell damage (31).
The nucleoprotein (p40) and phosphoprotein (p24) of the virus are most
abundantly synthesized during BDV infection and represent the main
targets for the immune system. Recently, we have shown that the
nucleoprotein is a major target for CTL in the Lewis rat model (32). In
this report, we describe the characterization and quantification of a
naturally processed RT1.Al ligand from the nucleoprotein of
BDV. This peptide is recognized by classical CD8+ T cells.
Virus and Experimental Animals--
The Giessen strain He/80 of
BDV was used for this study (33). Female Lewis rats were purchased from
the central breeding facilities of the Federal Research Center for
Viral Diseases of Animals in Tübingen. At an age of 5 weeks the
animals were infected intracerebrally in the left brain hemisphere with
0.05 ml of BDV corresponding to 5 × 103 focus-forming units.
Cell Lines and Cell Collection--
Skin cell cultures were
obtained from 2-week-old Lewis (LEW), Brown Norway (BN), and Louvain
(LOU) rats and cultured for more than 8 years in our laboratory (28).
F10 (Lewis astrocytes) cells were originally obtained from Dr. H. Wekerle, Munich. OX 18 hybridoma cells, secreting antibodies directed
against RT1.A were purchased from ATCC and cultured in our laboratory.
In addition, LEW and F10 cells were persistently infected with BDV
(BDV-LEW and BDV-F10), and persistent infection was controlled on a
routine basis by immunofluorescence or fluorescence-activated cell
sorter analysis. For peptide elution BDV-LEW or BDV-F10 cells were
cultured in a spin bottle system using CultisphereTM microcarrier
(Integra, Fernwald, Germany). This system allowed the production of
1-2 × 109 cells/liter. OX18 monoclonal antibody was
produced in 350-ml CELLINETM flasks (Integra, Fernwald, Germany).
Preparation of BDV-specific T Cells--
Lymphocytes from the
brains of BDV-infected rats were isolated by a method previously
described (34) and modified for the BDV infection of rats (28). Twenty
days after BDV infection rats were anesthetized with ketamine
hydrochloride and perfused with balanced salt solution. The brain
tissue was carefully homogenized through a stainless steel mesh and
collected in balanced salt solution containing collagenase D (0.05%),
trypsin inhibitor (TLCK; 0.1 µg/ml), DNase I (10 µg/ml), and
HEPES (10 mM). The cell suspension was stirred at room
temperature for 1 h and allowed to settle for 30 min. The
supernatant was pelleted at 200 × g for 5 min. The
pellet was resuspended in 10 ml of calcium-magnesium-free phosphate-buffered saline. Five ml of the suspension were layered on
top of 10 ml of a modified RPMI medium-Ficoll gradient and centrifuged
at 500 × g for 30 min. The pellet containing the
lymphocytes was resuspended in IMDM with 5% rat serum and 5% ConA
supernatant and cultured overnight. The next day, cells were counted
for further use.
In Vitro Cytotoxicity Assay and Peptide
Labeling--
Effector T cells were used in a concentration of 3 × 106 cells/ml or 106 cells/ml IMDM,
2% fetal calf serum. Persistently BDV-infected LEW (BDV-LEW) were
labeled with 0.2 mCi of 51Cr at 37 °C for 1 h,
washed three times with balanced salt solution, and used as target
cells. Dried HPLC peptide fractions were resuspended in a standard
volume of 150 µl of phosphate-buffered saline. For titration, 50 µl
of each fraction either undiluted or diluted 1:10 or 1:100 were
used to pulse 104 uninfected LEW cells in 50 µl of IMDM,
2% fetal calf serum for 90 min at 37 °C. Thereafter, 100 µl of
effector cells (effector to target ratio of 30:1 or 10:1) were added
and incubated for 10 h at 37 °C. Synthetic peptides were
dissolved in Me2SO in a concentration of 1 mg/ml.
For peptide titration, either 5 or 1 µl of the different peptides and
1:10 and 1:100 dilutions in a volume of 50 µl of IMDM, 2%
fetal calf serum were used to pulse 104 LEW cells in 50 µl of IMDM, 2% fetal calf serum for 90 min at 37 °C. Effector
cells were used as described above. For effector cell titration, the
standard peptide ASYAQMTTY was used in a concentration of 20 nM.
Extraction of MHC Class I Molecules and Isolation of Viral
Peptides from Infected Cells--
2.5 × 1010
virus-infected and uninfected LEW or F10 cells were resuspended in 200 ml of lysis buffer (phosphate-buffered saline, 10 mM CHAPS,
0.1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture tablets (Roche Molecular Biochemicals)) and disrupted using a handheld glass homogenizer and sonication. The suspensions were
stirred at 4 °C for 1 h before centrifugation at 4000 rpm for
10 min. The supernatants were spun in an ultracentrifuge at 40,000 rpm
for 1 h and passed through prefilters before loading onto
glycine-coupled cyanogen bromide-activated Sepharose 4B columns as a
preclearing step. The MHC I molecules were then purified by
immunoaffinity chromatography using monoclonal antibody OX18 coupled to
cyanogen bromide-activated Sepharose 4B. After elution of the
RT1.Al complexes using 0.1% trifluoroacetic acid
(pH 2), the eluted material was filtered through a Centricon 10 and concentrated to 0.5 ml by vacuum centrifugation.
Fractionation by HPLC--
Peptide separations were carried out
on a reversed-phase prepacked column (C2/C18, 2.1 × 100 mm;
Amersham Pharmacia Biotech) using the Amersham Pharmacia Biotech
SMART system. Samples were injected in a volume of 500 µl. The
following elution procedure was used: solvent A, 0.1% trifluoroacetic
acid in H2O; solvent B, 0.081% trifluoroacetic acid in
80% acetonitrile; 0-10 min, 10% B; 10-25 min, linear increase to
20% B; 25-45 min, 1%/min increase to 40% B; 45-55 min, 2%/min
increase to 60% B; 55-60 min, linear increase to 75% B; and 60-65
min, constant 75% B. The flow rate was 150 µl/min. Fractions were
collected by time fractionation (1-10 min, 450 µl/min; 10-65 min,
150 µl/min), and elution was monitored by measuring UV light
absorption at 214 nm in a continuous flow detector. Acetonitrile was
removed from eluted material by vacuum centrifugation before samples
were made up to a standard volume of 150 µl using phosphate-buffered
saline and stored at Epitope Prediction--
Potential RT1.Al-presented
peptides were selected by epitope prediction as described (35).
Briefly, nonamer peptides from the sequence of p40 (Swiss-Prot
accession number Q01552) and other Borna disease virus proteins
were selected using a matrix pattern suitable for the calculation of
peptides fitting to the RT1.Al peptide motif. The peptide
motif and epitope predictions are available on our web page,
where additional information can be obtained.
Synthetic Peptides--
Peptides were synthesized in an
automated peptide synthesizer 432A (Applied Biosystems, Weiterstadt,
Germany) following the Fmoc
(N-(9-fluorenyl)methoxycarbonyl)/tBu strategy. After
removal from the resin by treatment with trifluoroacetic
acid/phenol/ethanedithiol/thioanisole/water (90:3.75:1.25:2.5:2.5 by
volume) for 1 h or 3 h (arginine-containing peptides),
peptides were precipitated from methyl tert-butyl ether, washed once with methyl tert-butyl ether and twice with
diethyl ether, and resuspended in water prior to lyophilization.
Synthesis products were analyzed by HPLC (Varian Star, Darmstadt,
Germany) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (see below). Peptides of less than 80%
purity were purified by preparative HPLC.
Mass Spectrometry--
For MALDI-TOF MS 0.5 µl of sample was
mixed with 0.5 µl of dihydroxyacetophenone matrix (20 mg of
2,5-dihydroxyacetophenone, 5 mg of ammonium citrate in 1 ml of 80%
2-propanol) on a gold target and analyzed on a Hewlett-Packard G2025A
instrument (Hewlett-Packard, Waldbronn, Germany) at a vacuum of
10
Nanocapillary HPLC-MS and MSMS of synthetic and naturally processed
peptides were done as described (36) by coupling a reversed-phase HPLC
system (ABI 140D, Applied Biosystems) to a hybrid quadrupole orthogonal
acceleration time of flight tandem mass spectrometer (Q-TOF, Micromass,
Manchester, United Kingdom) equipped with an electron spray
ionization source. As a modification of the described setup,
loading of typical sample volumes of 100 µl was achieved by
preconcentration on a 300-µm × 5-mm C18
µ-precolumn (LC Packings, San Francisco, CA). A syringe pump (PHD
2000, Harvard Apparatus Inc., Holliston, MA), equipped with a gas-tight
100-µl syringe (1710 RNR, Hamilton, Bonaduz, Switzerland), was used
to deliver solvent and sample at a flow rate of 2 µl/min. A blank run
was performed prior to any HPLC-MS run to ensure that the system was free of any residual synthetic peptide.
For nanocapillary HPLC-MSMS experiments, fragmentation of the parent
ion was achieved at the given retention time by collision with argon
atoms. Q1 was set to the mass of interest ± 0.5 Da, and an
optimized collision energy was applied. Fragmentation was completed
after 60 s.
Recognition of Synthetic Peptides by BDV-specific T
Cells--
Peptide motifs and anchor residues of the
RT1.Al molecule have been published previously (15).
Therefore, the five entries of Borna disease virus proteins contained
in the Swiss Protein Database, release 39 (nucleoprotein,
phosphoprotein, matrix protein, glycoprotein, and the L-polymerase of
BDV), were screened for RT1.Al motifs using the data base
SYFPEITHI. Peptides optimal for presentation by RT1.Al are
nonamers carrying Phe or Tyr in position 3 and large hydrophobic residues in position 9. According to this prediction, several sequences
were synthesized from each viral protein (Table
I). Fibroblast cells from the Lewis rat
(LEW, RT1.Al) were loaded with peptides because no
TAP-deficient cell lines are available that express the
RT1.Al class I molecule.
To test whether the predicted peptides are recognized by BDV-specific
CD8+ T cells, Lewis rats were infected by the intracerebral
route, and 19 days later lymphocytes were isolated from the brain. As shown in Table I, these T cells recognized only 1 of the 16 predicted peptides when pulsed on LEW cells. The sequence ASYAQMTTY is located within the nucleoprotein of BDV.
After loading LEW cells with different concentrations of peptide, a
cytotoxicity assay using BDV-specific T cells as effector cells was
performed. As shown in Fig.
1A, the titration experiment indicated the highest specific cell lysis with 5 ng of peptide; half-maximal recognition was observed with 50 pg of peptide. When the
amino acid tyrosine at position 9 was changed to a glycine, the peptide
was still recognized to a lower extent (Fig. 1B), whereas after replacement of the tyrosine at position 3 by a glycine, cell lysis was only observed with the highest peptide concentration (Fig. 1C).
The RT1.Al restriction of BDV-specific T cells was
demonstrated by loading fibroblast cell lines from the Brown Norway rat (RT1.An) and from the Louvain rat (RT1.Au) with
peptide and using them as target cells. As shown in Table II, BDV-specific T cells are unable to
recognize peptide-labeled BN or LOU cells, whereas peptide-loaded LEW
cells as well as infected cells were killed. In addition, T cells from
BDV-infected rats were unable to kill YAC cells, demonstrating
the absence of NK cell activity (Table II). These results indicate that
the synthetic peptide ASYAQMTTY is recognized in combination with the
RT1.Al class I molecule by BDV-specific T cells from the
Lewis rat.
T Cell Recognition of Naturally Processed Peptides from
BDV-infected Cells--
Persistently BDV-infected LEW cells (2.5 × 1010) were lysed, and the RT1.Al complexes
were purified by immunoaffinity chromatography using the MHC class
I-specific monoclonal antibody OX18. The peptides were eluted from the
RT1.Al complexes and fractionated by HPLC. In control
experiments, RT1.Al-bound peptides from uninfected LEW
cells were eluted (Fig. 2B and
data not shown). Thereafter, uninfected LEW cells were incubated with
aliquots of the different HPLC fractions and were tested for
recognition by BDV-specific T cells. Significant BDV-specific lysis was
found with the HPLC fraction 24 and to a lower extent with fraction 23 (Table III, Fig. 2C). Similar
results were obtained when RT1.Al molecules from 2.5 × 1010 persistently BDV-infected F10 cells were
immmunoprecipitated and fractionated by HPLC (data not shown). As a
control, LEW cells were incubated with HPLC fractions 23 and 24 of the
peptide mixture eluted from a column that contained glycine instead of
the monoclonal antibody OX18. No lysis of target cells was observed,
indicating that HPLC fractions 23 and 24 of persistently BDV-infected
LEW cells contained peptides that are recognized by BDV-specific T cells (Table III). Furthermore, no specific lysis was found when target
cells were incubated with HPLC fractions of RT1.Al
molecules from 2.5 × 1010 uninfected LEW or F10 cells
(data not shown).
Coelution of the Synthetic Peptide ASYAQMTTY with the CTL-reactive
Fraction--
One µg of the synthetic peptide ASYAQMTTY was analyzed
by HPLC using identical conditions as for the separation of
RT1.Al ligands from BDV-LEW. As shown in Fig.
2A, an intense UV signal is visible in the HPLC profile at
fraction 24. Additional peaks, particularly in fractions 13, 28, and
47, result from medium contents, because the peptide had been
dissolved in IMDM before it was diluted in 0.1% trifluoroacetic acid.
Fraction 24 and fraction 23 were analyzed by MALDI-TOF mass
spectrometry. Only one m/z signal corresponding to the molecular mass of ASYAQMTTY (MH+ 1035) was detected,
indicating that ASYAQMTTY coeluted with the naturally processed peptide
recognized by BDV-specific T cells (data not shown). Furthermore, when
fractions 23 and 24 were used to label target cells, these cells were
lysed by BDV-specific T cells (Table III). Dilution experiments
indicated that the majority of ASYAQMTTY peptide eluted in fraction 24. Moreover, Table III suggests that in fraction 24 of the BDV-LEW, the
OX18 HPLC run contained less copies of the peptide than the
respective fraction of the HPLC run performed with the synthetic peptide.
Characterization and Quantification of the Natural
RT1.Al Ligand from Borna Disease Virus p40 on BDV-LEW
Cells--
Although CTL recognition after coelution experiments
indicated the presence of ASYAQMTTY in fraction 24 of the
RT1.Al ligand separation from infected LEW, the amino acid
sequence of this naturally processed peptide was confirmed by
nanocapillary liquid chromatography-MSMS analysis (Fig.
3). Comparison of liquid chromatography-MS signal intensities of the naturally processed peptide
and 2 pmol of coeluting synthetic peptide indicated that a total of 3.7 pmol of naturally processed ASYAQMTTY had been isolated from 2.5 × 1010 BDV-infected LEW cells (data not shown). This
corresponds to ~350 copies/cell, assuming an overall yield of 25%
after peptide extraction and HPLC (36).
In the present communication, we identified and characterized the
first rat MHC class I ligand derived from an infectious agent and
recognized by CD8+ T cells. The peptide was isolated from
BDV-infected cells upon MHC immunoprecipitation and purified by HPLC.
The peptide is recognized by BDV-specific T cells. Furthermore,
following peptide prediction, a synthetic peptide is recognized in
combination with the RT1.Al class I molecule of the Lewis
rat by BDV-specific T cells. Replacing an amino acid either in position
3 or position 9 results in clearly lower lytic activity, where
position 3 seemed to be more decisive for an efficient binding of the
peptide to MHC class I than did position 9. The peptide is located
within the nucleoprotein (p40) of the virus. After HPLC fractionation
of the synthetic peptide ASYAQMTTY, we found that this synthetic
peptide coelutes with the naturally processed peptide recognized by
BDV-specific T cells. Amino acid sequence by nanocapillary HPLC-MSMS
analysis confirmed that ASYAQMTTY is also a naturally processed
peptide. Furthermore, after quantification of the peptide, we
were able to show that ~350 copies of this peptide are complexed with
MHC class I molecules on the surface of a BDV-infected cell.
The knowledge of antigen processing and presentation in rats is
fragmentary compared with what is known in humans and mice. For the
laboratory rat (R. norvegicus) only the peptide motifs of
RT1.Al, RT1.Au, RT1.Ac, and
RT1.Aa are known, showing a restricted preference for
peptides of 9-12 amino acids (14-16, 37). This is similar to
preferences of MHC class I molecules in humans and mice. Nevertheless,
there are differences in antigen processing and presentation of rats
compared with humans and mice. The rat, as a unique feature, has two
functionally distinct allelic forms of TAP, TAP-A and TAP-B, which have
different peptide transport specificities (13). Peptides used in the
present study that were presented by RT1.Al are known to be
linked to TAP-A, which efficiently transports peptides with aromatic C
termini (38, 39). Because RT1.Al does not have an acidic
F pocket, the peptide ASYAQMTTY, probably transported by TAP-A,
fits perfectly into the RT1.Al class I molecule. Although
some information about naturally processed self- or allo-peptides from
the rat exists, no ligand from viruses or intracellular bacteria has
been reported so far. Recently, Stevens et al. (40)
described the first RT1.Ac class I allogenic NK ligand
after designing synthetic peptides based on the published binding motif
for RT1.Ac and the identification of naturally presented
peptides by RT1.Al on rat splenocytes. The present article
describes the first classical CTL epitope encoded by a virus.
Borna disease virus is found in a wide variety of mammals including man
(reviewed in Ref. 19). The best investigated experimental model of
Borna disease, a virus-induced immune-mediated encephalomyelitis, is
the infection of the Lewis rat. The knowledge of a defined CTL epitope
of BDV will help to further characterize the immunopathological mechanisms in more detail. An earlier study showed that only target cells infected with a recombinant vaccinia-BDVp40 construct were recognized by BDV-specific CTL, whereas target cells infected with
vaccinia virus carrying the phosphoprotein, the matrix protein, or the
glycoprotein were not recognized (32). Because the peptide ASYAQMTTY is
recognized by CTL most efficiently, one might assume that this peptide
represents an immunodominant trait. However, we cannot exclude the
existence of other, subdominant, nucleoprotein-specific CTL epitopes.
Because no NK cell activity was found in brain lymphocyte preparations,
NK-specific killing directed against the peptide ASYAQMTTY can be
excluded. This finding is supported by an earlier report in which
CD8+ T cell-mediated, MHC class I-restricted lysis of
BDV-infected target cells, but no killing of NK-sensitive YAC
cells, was found (28).
The quantification of the natural RT1.Al ligand ASYAQMTTY
from persistently BDV-infected cells showed that ~350 copies/cell were present. This copy number is similar to those reported from other
viral epitopes associated with MHC class I molecules from humans and
mice (41). In persistently BDV-infected cells, only a very few
infectious viral particles can be found (42, 43). Because BDV is a
negative-stranded RNA virus, one might speculate that high copy numbers
of p40 are required for RNA stabilization and consequently virus
replication. Therefore, our data provide additional information for the
biology of BDV and a better understanding of the poorly understood
mechanism of replication. MHC-restricted cytotoxic T cells recognize
virus-specific peptides in combination with MHC class I. This can take
place relatively early after infection in the absence of an infectious
virus (44). Recently it was shown that translation of RNA by ribosomes
into protein can result in defective ribosomal products, leading to an
early recognition of the virus-infected cell by the immune system,
whereas the foreign proteins are still being produced (45, 46). These
findings support the earlier investigations by Zinkernagel and Doherty (1, 44) and also suggest that the numbers of copies found for a
peptide must not correlate with the amount of protein made and needed
for virus replication. On the other hand, because the gene encoding for
the nucleoprotein is located at the 3' end of the antigenome (open
reading frame I), and therefore viral transcription and
translation of this protein occur very early, the nucleoprotein is a
good candidate for an early immunodominant CTL response of the host
against BDV. This hypothesis is supported by the findings that p40 is
the first BDV-specific protein detectable in infected cells and tissue
and that BDV-specific CD8+ T cells are directed against the
nucleoprotein (32, 47).
During the last 15 years BDV was repeatedly found in patients with
psychiatric disorders (20, 21, 23). Nevertheless, it is not clear if
BDV is the causative agent of these disorders or if it is simply a
secondary infection. Antibodies in humans were found to be
predominantly directed against the nucleoprotein, the phosphoprotein,
and the matrix protein (25, 48, 49). The role of the cellular immune
response against BDV in man is still unknown. With our data obtained in
the BDV model system and with the help of epitope prediction and
transgenic mouse models, one might be able to define BDV-specific
HLA-restricted CTL epitopes to investigate a possible CTL response in man.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain and the
2-microglobulin in association with a peptide derived
from cytoplasmic proteins after proteolytic cleavage by proteosomes
(5). The resulting peptides are transported into the lumen of the
endoplasmic reticulum with the help of a heterodimeric transporter
associated with antigen presentation (TAP) (6, 7). The
-chain,
2-microglobulin, and peptide are assembled in the
endoplasmic reticulum, and the mature MHC class I molecule migrates
through the Golgi to reach the cell surface. Most of the allelic
polymorphism of the
-chain is confined to the
1-
2 domains, which form a membrane-distal groove, and specific binding is defined by key amino acids within the
peptide, referred as anchor residues (reviewed in Ref. 8). In contrast to humans and mice, in the laboratory rat
(Rattus norvegicus) the function of CD8+ T cells
is poorly understood, and only a few experimental models of
intracellular infectious agents are available to analyze T cell functions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. For the coelution experiments,
1 µg of synthetic ASYAQMTTY diluted in 0.1% trifluoroacetic acid was
injected in a total volume of 500 µl and separated using the same
conditions as described above.
6 torr (1 torr = 133 pascals). For
signal generation, 50-150 laser shots were added up in the single shot mode.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Recognition of synthetic peptides by BDV-specific CD8+ T cells
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Fig. 1.
BDV-specific T cells were isolated from the
brains of BDV-infected rats (see "Experimental Procedures").
LEW cells were used as target cells and labeled with peptide ASYAQMTTY
(A), peptide ASYAQMTTG where position 9 was replaced by a
glycine (B), or ASGAQMTTY where position 3 was replaced by a
glycine (C) in different amounts as indicated. An effector
to target ratio of 30:1 was used. After 10 h 50 µl of the
supernatant were harvested and counted in a Packard gamma counter, and
percent specific lysis was determined. Spontaneous release in this
experiment was 28%. The experiment with peptide A was repeated four
times; experiments with peptide B and C were done twice.
MHC restriction of the BDV-specific peptide ASYAQMTTY
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Fig. 2.
Coelution of synthetic ASYAQMTTY
(A) and CTL-reactive fractions of natural ligands
extracted from BDV-infected cells (B).
BDV-specific CD8+ T cells recognized identical fractions of
both HPLC separations after loading LEW target cells with HPLC
fractions (C shows recognition of natural ligands). The
effector to target ratio was 20:1. After 10 h, 50 µl of the
supernatant were counted in a Packard gamma counter, and percent
specific lysis was determined. Spontaneous lysis was
26%.
Recognition of HPLC fractions by BDV-specific T cells
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Fig. 3.
Liquid chromatography-MSMS analysis of the
CTL-reactive fraction 24. Low energy collision-induced
dissociation spectrum recorded on ions having a
m/z of 1035.5 coeluting with the synthetic
peptide ASYAQMTTY. Only peaks corresponding to b, a, and immonium ions
are labeled.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank H. G. Rammensee and R. M. Zinkernagel for critical reading of the manuscript and Patricia Hrstic for expert technical assistance.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grants Pl 256/1-1 (to O. P. and L. S.) and Sti 72/2-2 (to L. S. and O. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Institut für Immunologie, Bundesforschungsanstalt für Viruskrankheiten der Tiere, Paul-Ehrlich-Str. 28, 72076 Tübingen, Germany. Tel.: 49 7071 967 254; Fax: 49 7071 967 105; E-mail: oliver.planz@tue.bfav.de.
Present address: Universitätsspital Zürich, Dept.
of Pathology, Inst. of Experimental Immunology, Schmelzbergstr. 12, CH-8091 Zürich, Switzerland.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009889200
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ABBREVIATIONS |
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The abbreviations used are: MHC, major histocompatibility complex; CTL, cytotoxic T-lymphocyte; TAP, transporter associated with antigen presentation; BDV, Borna disease virus; LEW, Lewis; IMDM, Iscove's modified Dulbecco's medium; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; BN, Brown Norway; LOU, Louvain.
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