This study describes the construction of soluble major histocompatibility complexes consisting
of the mouse class I molecule, H-2Db, chemically biotinylated
2 microglobulin and a peptide
epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I
complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific
cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific
for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the
cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8+ T
cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the
study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.
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Introduction |
The ability to clear infections with noncytopathic viruses is predominantly attributed to CD8+ CTLs.
CTLs recognize infected cells via an interaction between TCRs and their corresponding ligands, class I MHC molecules (1). MHC class I molecules are expressed on the cell
surface in association with self or pathogen-derived peptides that are generated intracellularly by proteolytic degradation of the parent proteins (2). After recognition of an
infected cell, naive CTLs become activated, proliferate, and
attain not only the ability to lyse infected cells, but also the
ability to produce IFN-
(3). Although IFN-
may have a
direct antiviral effect (4, 5), it has also been shown to improve the efficiency of antigen presentation by class I molecules (6) thereby promoting the induction of a CTL response and improving the efficiency with which CTL can recognize their infected targets.
CTLs have been shown to be essential for the recovery
of mice from the acute phase of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV; reference 10). So far, it has not been possible to follow the kinetics of appearance and disappearance of antigen-specific
effector CTLs during the acute phase of both low- and
high-dose LCMV infections in non-TCR-transgenic mice. A recent study by Altman et al. (11) described a method,
using tetrameric soluble MHC class I-peptide complexes,
for the identification of antigen-specific CD8+ cells in the
PBMCs of HIV-infected humans. This study describes an
adaptation of this method for the identification of antigen-specific CD8+ cells in B6 (H-2b) mice infected with
LCMV. In this case, soluble peptide-MHC complexes were generated using the mouse class I heavy chain Db,
chemically biotinylated human
2 microglobulin (
2M)
and the LCMV peptide epitope glycoprotein (GP)33-41
(GP33-KAVYNFATC). Fluorescence-labeled tetrameric
complexes were subsequently produced by mixing the biotinylated complexes with phycoerythrin-labeled neutravidin. Peptide GP33-41 (GP33) was used for the purposes of
this study since, after LCMV-WE infection of C57BL/6
mice, most CTL activity (~50-60%) is directed towards
this epitope. Two other epitopes, defined by residues 276-
286 of the viral glycoprotein (GP276) and residues 396-
404 of the viral nucleoprotein (NP396), represent 10-20
and 20-30% of the total CTL activity, respectively (12).
The tetrameric class I-peptide complexes, which stained
CTLs specifically, were used to follow the fate of GP33-specific CD8+ T cells in mice during the acute phase of
LCMV infection. This study demonstrates the accummulation of stained virus-specific CTLs in the CSF and spleens
of mice after intracranial infection with a substrain of
LCMV-WE called LCMV-DOCILE, and in the spleens of
mice infected intravenously with the same virus. In both
cases, the accummulation of GP33-specific CTLs, after
both low- and high-dose infection with LCMV-DOCILE,
was monitored in relation to the capacity of the cells to
produce IFN-
; to mediate cytotoxic activity, and to mediate virus clearance.
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Materials and Methods |
Mice.
C57BL/6 (H-2b), 318 TCR transgenic mice (18), and
perforin-deficient mice (PKOB; reference 10) were obtained from
the Institut f ür Zuchthygiene (Tierspital Zürich, Switzerland). All
mice were kept in a specific pathogen-free mouse housing facility.
Peptides.
The LCMV peptides GP33-41, nucleoprotein (NP)
396-404, and GP276-286 were purchased from Neosystem Laboratoire (Strasburg, France).
Virus.
The LCMV-DOCILE strain was a gift from C. Pfau
(C. Pfau, Rensselaer Polytechnical Institute, Troy, NY) and was
grown using Madin darby canine kidney (MDCK) cells. Recombinant vaccinia virus (rVV) expressing the GP of vesicular stomatitis virus (VSV) serotype Indiana (rVVINDG) has been described
previously (19). This virus was grown in bovine skin cells (BSC)-1.
All virus stocks were stored at
70°C. C57BL/6 mice were intravenously infected either with 200 µl of LCMV at 103 PFU/ml
or with 200 µl of LCMV at 107 PFU/ml. Intracranial injections
were carried out using 30 µl of LCMV at 103 or 107 PFU/ml and
20 µl of rVVINDG at 105 PFU/ml.
Cells and Media.
Cultures of the methylcholantrene-induced
murine fibroblast line, MC57, were maintained in MEM supplemented with 5% fetal calf serum, penicillin-streptomycin, and
L-glutamine. The Rauscher virus-transformed mouse T cell line,
RMA-S (20), and the human Tap-defective cell line, T2 (21)
transfected with H-2Db, were maintained in RPMI supplemented with 10% fetal calf serum, penicillin-streptomycin, and
L-glutamine.
Detection of Virus-specific Cytotoxic T Cells.
Single cell suspensions were prepared from the spleens of mice infected intravenously or intracranially with the indicated doses of LCMV at various time points. Cells were resuspended in complete MEM and
used directly in cytotoxicity assays. NK cells were induced by intravenous injection of 100 µg poly-IC 24 h before the spleen
cells were tested using the NK-sensitive YAC-1 cell line as target
cells. The target cells used were either MC57 cells that had been
infected by incubation with 0.1 PFU LCMV/cell 48 h before the
experiment or MC57 cells pulsed with 100 µl of peptide (100 ng/ml). Cells were resuspended in complete MEM and used directly in cytotoxicity assays. Cytotoxicity assays were carried out
as described previously (22, 23).
Generation of Polyclonal CTL Lines.
Spleen cell suspensions were
prepared from mice that had been intravenously infected with
200 PFU LCMV-WE at least 3 mo previously. Cells were plated
at 4 × 106 cells/well (24-well plates) in 1 ml IMDM/well supplemented with 10% fetal calf serum, penicillin-streptomycin, 2-mercaptoethanol, and 10% Con A supernatant. The cultures were supplemented with 1 ml of peptide-pulsed irradiated RMA-S cells at a
concentration of 4 × 105/ml. Before irradiation, RMA-S cells
were incubated with 100 µl of peptide at a concentration of 10 ng/ml for 1 h at 37°C before extensive washing to remove any
unbound peptide. Cultures were restimulated at 14-d intervals
using irradiated peptide-pulsed RMA-S cells as APCs at a responder/APC ratio of 10:1. These CTL lines were found to be of
a single specificity after three rounds of restimulation.
Virus Titration.
LCMV titers in spleens were determined as
previously described (24).
Isolation of Cerebrospinal Fluid.
Cerebrospinal fluid was isolated from mice which had been infected after intracranial inoculation with LCMV-DOCILE as described previously (25, 26).
Protein Expression and Refolding.
The H-2Db expression vector was constructed by PCR of the gene fragment encoding the
1,
2, and
3 domains and the first two amino acids of the transmembrane domain (residues 1-276) using the primers 5'-CATATGACATATGGGCCCACACTCGATGCGGTATTTC-3' and
5'-CATATGAAGCTTTTATCAAGGCTCCCATCTCAGGGT-3'. The resulting fragment was cut with the restriction enzymes
NdeI and HindIII (New England Biolabs, Beverly, MA) and
cloned into the expression vector pGMT7 (a pET derivative; reference 27). The expression plasmid was transformed into the Escherichia coli strain BL21 (DE3) pLysS (Novagen, Madison, WI)
and grown at 37°C in Luria-Bertani medium containing 100 µg/
ml ampicillin (Sigma Chemical Co., St. Louis, MO). Protein expression was induced at midlog phase (A600 = 0.6) with 0.5 mM
isopropyl-
-D-thiogalactosidase (IPTG). After 5 h, the cells were
harvested and lysed by an overnight freeze thaw step (
80°C).
Inclusion bodies were isolated as follows. The lysed pellet from 1 liter of culture was resuspended in 40 ml of 25% sucrose in 10 mM Tris (pH 8.0), 1 mM EDTA, 1 mM PMSF, and 10 mM
dithiothreitol (DTT) and sonicated until no longer viscous. The
solution was then centrifuged at 18,000 rpm for 30 min before the supernatant was discarded and the pellet resuspended in 50 ml
of 50 mM Tris, pH 8.0, 25% sucrose, 1% Nonidet P-40, 0.5% sodium deoxycholate, 5 mM EDTA, and 2 mM DTT. The insoluble material was again recovered by centrifugation at 18,000 rpm
for 30 min and resuspended in 50 ml of 25 mM Tris (pH 8.4), 2 mM DTT, 2 M NaCl, and 2 M urea. After centrifugation as
above, the washed inclusion body pellet was resuspended in 5 ml
of 20 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM PMSF, and
stored at
20°C. The inclusion bodies were solubilized in 8 M
urea at 4°C for 6 h immediately before refolding.
2M was produced as described above from the vector pHN
2M (28). After
solubilization of the
2M inclusion bodies in 6 M guanidinium
HCl (pH 8.2), the protein was biotinylated using a fivefold molar
excess of N-hydroxysuccinimide biotin (Sigma Chemical Co.).
After a 30-min incubation at room temperature and a further 1-h
incubation on ice, the biotinylation reaction was stopped by adding NH4Cl to a final concentration of 10 mM. The biotinylated
2M was dialyzed against 6 M guanidinium HCl to remove any
free biotin and was subsequently used in refolding reactions. A dilution method of refolding was used to produce specific H-2Db-
peptide complexes (29). In brief, denatured biotinylated human
2M and H-2Db heavy chain (>1 mg/ml in 8 M urea) and peptide
were diluted into refolding buffer (0.4 M L-arginine, 0.1 M Tris,
pH 7.5, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, and 0.5 mM PMSF) to a final concentration of
0.76 µM heavy chain, 1.15 µM
2M, and 7.6 µM peptide. Refolding was carried out with stirring at 4°C for 36-48 h. The refolding solution was then concentrated using an Amicon (Millipore, Watford, UK) stirred cell and centriprep (MW cutoff:
10,000) and purified by gel filtration using a Superdex-75 column
(Pharmacia, Piscataway, NJ) in 20 mM Tris-HCl (pH 7.5) and
150 mM NaCl on an FPLC system.
Construction of Tetrameric Class I-Peptide Complexes.
Phycoerythrin-labeled neutravidin (Molecular Probes Inc., Eugene, OR) was
mixed stepwise with biotinylated H-2Db complexes containing
peptide GP33-41 at a 1:4 molar ratio. The tetrameric complexes
were subsequently concentrated using a centriprep (MW cutoff:
100,000) to 1 mg MHC class I complex/ml. Aliquots of 2 × 105
cells were stained using 50 µl of Tetramer. Approximately
200,000 cells were analyzed by flow cytometry.
Flow Cytometry.
Single cell suspensions were prepared from
the spleens of LCMV-infected or uninfected control mice. 2 × 105 cells were stained for 30 min with tricolor-conjugated anti-CD8 (Caltag Labs., South San Francisco, CA). After washing in
PBS containing 2% FCS and 0.5 mM EDTA (PBS/FCS/EDTA),
the cells were stained using 50 µl of the solution containing tetrameric class I-peptide complexes. After a 45-min incubation, the
cells were washed twice, resuspended in PBS/2% FCS/0.5 mM
EDTA (FACS buffer), and analyzed by flow cytometry (FACSCAN®; Beckton Dickinson, Mountain View, CA) using Cellquest
software.
Intracellular cytokine staining was carried out by incubating
106 cells/ml for 2 h at 37°C in the presence of ionomycin (1 µM) and PMA (20 ng/ml). Subsequently, the cells were incubated for a further 2 h in the presence of 2 µM monensin. After washing with FACS buffer, the cells were stained as described above using tricolor-conjugated anti-CD8 antibodies and tetrameric class I-peptide complexes. Subsequently, the cells were fixed in 100 µl PBS
containing 2% (wt/vol) paraformaldehyde and permeabilized using PBS containing 1% FCS, 0.1% (wt/vol) sodium azide, and
0.1% (wt/vol) saponin (permeabilization buffer). Permeabilized
cells were stained with FITC-conjugated anti-IFN-
antibodies
(Pharmacia, St. Albans, UK). The stained cells were washed twice
in permeabilization buffer, resuspended in FACS buffer, and analyzed by flow cytometry. In all cases, staining and washing of the
cells was carried out at 4°C.
 |
Results and Discussion |
TCR Staining Using Tetrameric Class I-Peptide Complexes.
GP33 tetramer staining of GP33-specific CD8+ T cells was
examined in spleen cells from a 318 TCR transgenic mouse.
Approximately 40-60% of T cells in the 318 mouse express
the transgenic TCR designated P14 (V
2V
8.1), which
recognizes H-2Db in association with peptide GP33. 318 spleen cells were stained with anti-CD8 antibodies and with
GP33 tetramers or antibodies specific for V
2. As shown in
Fig. 1, B and D, the percentage of CD8+ cells that stained
positive with GP33 tetramers correlated well with the percentages obtained using the transgenic TCR-specific antibody. In contrast, only 3.7% of CD8+ cells recovered from
a naive B6 mouse stained positive with GP33 tetramer (Fig.
1 C). GP33 tetramers were also used to stain polyclonal CD8+ CTL lines that had been generated after peptide restimulation of spleen cells from LCMV-immune mice.
These CTL lines had been maintained in culture for 2 mo
by weekly restimulation with RMA-S cells pulsed with either peptide GP33-41, GP276-286, or NP396-404. Cell
staining was carried out 14 d after the last restimulation. As
shown in Fig. 1 E, although GP33 tetramers stained the
CTL line specific for peptide GP33, no staining of CTL
lines specific for peptides GP276 and NP396 was observed.
These experiments demonstrate the specificity and potential usefulness of tetrameric class I-peptide complexes in the
identification of antigen-specific T cells. Tetrameric class I
complexes were used to stain GP33-specific T cells in the
CSF of mice infected intracranially 7 d before with 30 PFU of LCMV-DOCILE. Intracerebral infection with LCMV
induces a lethal CD8+ T cell-dependent and perforin-mediated immunopathology (10). In B6 mice, the specific
inflammatory response begins by days 5-6 after infection
and reaches ~104 infiltrating cells/µl of CSF on days 6-7.
The results, shown in Fig. 2 B, indicate that ~24% of
CD8+ cells recovered from CSF on day 7 after infection
with LCMV-DOCILE were specific for peptide GP33. As
a negative control, B6 mice were infected intracranially
with 2 × 103 PFU of rVVINDG. Although similar numbers of cells were recovered from the CSF of these mice,
no significant staining of the cells was observed using the
GP33 tetramers (Fig. 2 A).

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Fig. 1.
Staining of GP33-specific H-2Db-restricted TCR transgenic T cells and polyclonal LCMV-specific CTL lines with GP33 tetramers. Spleen
cells from a naive B6 mouse and a 318 TCR transgenic mouse were stained with monoclonal antibodies specific for CD8 and the TCR gene segment
V 2 (A and B) or with anti-CD8 and GP33 tetramers (C and D). CD8+ CTL lines specific for H-2Db-restricted LCMV peptide epitopes, GP33-41,
NP396-404, and GP276-286 were stained using GP33 tetramers 2 wk after in vitro restimulation with peptide-pulsed spleen cells (E).
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Fig. 2.
Staining of GP33-specific Db-restricted CD8+ cells isolated
from the CSF of mice infected intracranially with rVVINDG or LCMV-DOCILE. Cells isolated from the CSF of mice infected intracranially with
2 × 103 PFU of rVVINDG (A) or 30 PFU of LCMV-DOCILE (B) were
stained with anti-CD8 antibodies and GP33 tetramers. The histograms
show staining of live CD8+ cells with GP33 tetramers.
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Staining of GP33-specific CD8+ Cells after Intravenous Inoculation of B6 Mice with LCMV-DOCILE.
Spleen cells from
mice infected intravenously with either a low dose (2 × 102 PFU) or a high dose (2 × 106 PFU) of LCMV-DOCILE
were stained using anti-CD8 antibodies and GP33 tetramers at days 3, 6, 9, and 15 after infection. After low-dose
infection with this virus, GP33-specific CD8+ cells were
barely detectable at day 6 after infection (Fig. 3 A), but had
increased significantly in number by day 9 after infection to
comprise ~1 in 3 CD8+ T cells. On day 9, viral titers were
no longer detectable in the spleens of the infected mice
(Fig. 4). After infection with a high dose of LCMV-DOCILE,
GP33-specific CD8+ cells comprised almost 1 in 2 CD8+
T cells at day 3 after infection, but had already decreased in number by day 6 after infection and were barely detectable
at day 9 after infection (Fig. 3 B). In contrast to the situation observed after low-dose infection and despite the rapid
induction of GP33-specific cells, these cells were not able
to control the infection since virus titers continued to increase in the spleens of these mice after the time point at
which GP33-specific cells were no longer detectable (Fig.
4; reference 30).

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Fig. 3.
CD8+ and GP33-specific Db-restricted CD8+ cells isolated
from the spleens of mice infected intravenously with LCMV-DOCILE.
Cells isolated from the spleens of mice infected intravenously with low or
high dose of LCMV-DOCILE were stained with anti-CD8 antibodies
and GP33 tetramers. The graphs describe data collected from histograms
generated as described in Fig. 2. Solid bars, the percentage of CD8+ cells
in the spleens of individual mice at four time points during the acute
phase of LCMV infection; open bars, the percentage of CD8+ cells that
also stained with GP33 tetramers. The results are representative of two independent experiments carried out using groups of two mice. The number of GP33-specific CD8+ cells (× 105) are shown in brackets above
each bar.
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Fig. 4.
Cytotoxic activity and virus titers in mice infected intravenously with LCMV-DOCILE. LCMV-specific CTL activity was measured using spleen cells from mice that had been infected intravenously
with a low (A-D) or high dose (E-H) of LCMV-DOCILE. Spleen cells
were tested for lysis of normal MC57 cells ( ) or MC57 cells that had either been pulsed with peptide GP33 ( ) or which had been infected with
LCMV-DOCILE ( ). Virus titers, measured as PFU in the spleen (Spl)
and thymus (Thy) of each mouse are shown in the upper right-hand corner of each panel. 200 PFU represents the detection limit of the assay.
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GP33-specific CTL Responses in B6 Mice Infected Intravenously with LCMV-DOCILE.
To establish if a correlation
exists between the expansion of GP33-specific cells observed by flow cytometry and GP33-specific cytotoxicity,
direct CTL assays were carried out using spleen cells from
the mice described above. CTL activity mediated by antigen-specific CD8+ cells is usually difficult to measure at
day 3 after infection since LCMV induces, at this early time
point, strong NK cell activity that lyses both antigen-pulsed
and unpulsed target cells. To circumvent this problem, direct CTL assays were carried out by adding a twofold excess of unlabeled NK-sensitive target cells (YAC-1 cells) to assay wells as cold-target competitors. Inclusion of cold
YAC-1 cells abrogated lysis of chromium-labeled MC57
target cells by NK cells from the spleens of mice that had
been injected 48 h previously with poly-IC (data not
shown). No inhibition of antigen-specific CTLs was observed when YAC-1 cells were included as cold-target competitors in a direct CTL assay using spleen cells from
mice that had been infected 8 d before with LCMV (data
not shown). Thus, antigen-specific CTL activity in spleen
cells from mice infected 3 d previously with LCMV was
measured in the presence of cold YAC-1 cells. Although
only low levels of specific killing were observed using
spleen cells from mice infected with a low dose of LCMV-DOCILE, strong cytotoxic activity was evident in the
spleens of mice that had been infected with a high dose of the same virus (Fig. 4, A and E). The situation was reversed
at days 6 and 9 when CTL activity gradually increased in
mice infected with a low dose of LCMV-DOCILE, but
declined to low levels in those mice that had been infected
with a high-dose of virus (Fig. 4, B-G). Despite the observation that GP33 tetramer+/CD8+ T cells had fallen to
background levels in both low- and high-dose infected
mice by day 15 after infection, GP33-specific CTL activity was still detectable using spleen cells from mice that had
been infected with a low dose of LCMV-DOCILE. No
CTL activity was measurable in mice infected with a high
dose of the same virus (Fig. 4, D and H). After in vitro restimulation, cytotoxic activity was readily detectable in
spleen cells from mice that had previously eliminated infection with LCMV-DOCILE and these mice remained protected against any further challenge with the same virus
(30, and data not shown). Infection with a high dose of
LCMV-DOCILE resulted in a very rapid induction of virus-specific CTLs. This response peaked both in terms of
cell numbers and in cytotoxic activity before virus titers began to decline. Thus, as has been previously shown, high-dose infection with a rapidly replicating strain of LCMV
resulted in an overwhelming burden of antigen causing an
early and complete induction of the CD8+ effector population (30). Since terminally differentiated effector cells are
thought to die within 2-3 d, the effectiveness of the CTL
stimulated after infection is too short lived to have a significant effect on the spread of virus. As has been reported
previously, no cytotoxic T cell activity could be measured
after in vitro restimulation of spleen cells isolated from persistently infected mice (30). This may reflect both deletion
of the LCMV-specific T cells in the periphery and the continuous deletion of differentiating specific thymocytes in
LCMV-infected thymi, which prevents repopulation of the
peripheral T cell pool.
Production of IFN-
by GP33-specific T Cells after Intravenous Infection of B6 Mice with LCMV-DOCILE.
CD8+ T
cell populations in mice infected intravenously with LCMV-DOCILE were examined for their capacity to produce
IFN-
. Spleen cells from naive B6 mice and mice that had
been infected intravenously 8 d previously with a low dose
of LCMV-DOCILE were stimulated with PMA and ionomycin, permeabilized, and stained with anti-IFN-
antibodies. Expression of IFN-
was analyzed by flow cytometry. Fig. 5 shows that after LCMV infection, CD8+ T cells
(C), including those that are GP33 specific (D), exhibit an
elevated capacity to produce IFN-
after stimulation with
PMA and ionomycin when compared to stimulated (B)
spleen cells recovered from naive mice. Subsequently, CD8+
cells recovered from the spleens of mice that had been infected 3, 6, 9, or 15 d previously with either a low- or
high-dose of LCMV-DOCILE were stained with GP33
tetramers and examined for their capacity to produce IFN-
.
The results, shown in Fig. 6, indicate that following both
low- and high-dose infection with LCMV-DOCILE, the
capacity of tetramer+/CD8+ cells to produce IFN-
exhibits
the same kinetics as cytotoxic activity (compare to Fig. 4).

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Fig. 5.
Intracellular cytokine staining of CD8+ cells isolated from
naive and LCMV-DOCILE-infected B6 mice. Spleen cells isolated from
naive mice were stained directly (A) or after stimulation with PMA and
ionomycin (B) using anti-CD8 and anti-IFN- antibodies. Spleen cells
recovered from mice infected 8 d before with a low dose (2 × 102 PFU
intravenously) of LCMV-DOCILE were stimulated with PMA and ionomycin and subsequently stained with anti-CD8 antibodies, GP33 tetramers,
and anti-IFN- antibodies. C represents IFN- staining of the gated
CD8+ cell population, whereas D represents IFN- staining of the gated
CD8+/tetramer+ cell population. The percentage of gated cells that express IFN- is shown in each panel.
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Fig. 6.
Intracellular cytokine staining of CD8+ cells isolated from
LCMV-DOCILE-infected B6 mice. Spleen cells recovered from mice
that had been infected intravenously with either low- (2 × 102 PFU) or
high-dose (2 × 106 PFU) LCMV-DOCILE were stimulated with PMA
and ionomycin and subsequently stained with anti-CD8 antibodies, GP33
tetramers, and anti-IFN- antibodies. Subsequent FACS® analysis was
carried out as described for Fig. 5. Solid bars, the percentage of CD8+/
GP33 tetramer+ cells (from the experiment shown in Fig. 3) recovered
from the spleens of each mouse; open bars, the percentage of CD8+/GP33
tetramer+ cells that express IFN- .
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Virus-specific CTL Activity after Intracranial Inoculation with
LCMV.
Intracranial inoculation of immunocompetent
wild-type mice with a low dose (30 PFU) of LCMV results
in the induction of lethal choriomeningitis (31). The immunopathology observed in these mice correlates directly
with the cytotoxic action of virus-specific CD8+ T cells
since although mice lacking the perforin gene remain persistently infected with LCMV, they do not die of disease
(10). As shown in Fig. 7, A, B, E, and F, GP33-specific
CD8+ T cells were readily detectable in the spleen and
CSF of mice 7 d after infection with a low dose of LCMV-DOCILE. Direct CTL assays carried out using spleen cells
from these mice show that although these cells exhibit
strong cytotoxic activity (Fig. 8 A), the infection is not
cleared; the mice develop choriomeningitis and usually die
within the next 48 h.

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Fig. 7.
Staining of GP33-specific
CD8+ cells isolated from the CSF and
spleens of mice infected intracranially
with LCMV-DOCILE. Cells isolated
from the CSF and spleens of mice infected with low (A, B, E, and F) or high
(C, D, G, and H) doses intracranially with
LCMV-DOCILE were stained with anti-CD8 antibodies and tetramers. (Left)
Staining of live cells with anti-CD8 antibodies; (right) gated CD8+ cell population
stained with GP33 tetramers. The percentage of CD8+ cells (A, C, E, and G)
and the percentage of CD8+/tetramer+
cells (B, D, F, and H) is shown in each
panel. The mean fluorescence of splenic
GP33 tetramer-stained CD8+ T cells is
shown in brackets in F and H. The results
are representative of two independent
experiments carried out using groups of
two mice.
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Fig. 8.
Cytotoxic activity and virus titers in mice infected intracranially with LCMV-DOCILE. LCMV-specific CTL activity was measured
using spleen cells from mice that had been infected intracranially with a
low (A) or high (B) dose of LCMV-DOCILE. Spleen cells were tested for
lysis of normal MC57 cells ( ), MC57 cells that had either been pulsed
with peptide GP33 ( ), and MC57 cells which had been infected with
LCMV-DOCILE ( ). Virus titers, measured per spleen and brain of each
mouse, are also shown.
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Mice infected intracranially with a 1,000 times higher
dose (3 × 104 PFU) of LCMV-DOCILE also show signs of
disease between days 7 and 9 after infection. They, however, recover from transient disease and survive the infection, even though they remain persistently infected with
LCMV. This observation has been attributed to the exhaustion of virus-specific CTLs caused by the presence of
excessive antigen. In keeping with this hypothesis, staining
of lymphocytes isolated from the spleen and CSF of mice 7 d
after intracranial infection with a high dose of LCMV
showed that although relatively few CD8+ cells stained
GP33 tetramer positive (Fig. 7, D and H), TCR expression was reduced when compared to cells isolated from the CSF
of mice infected with a low dose of the same virus (compare mean fluorescence, parentheses, Fig. 7, F and H).
Downregulation of TCR expression, which has been
shown to occur after sustained contact between T cells and
their corresponding antigen (32) correlated with very
little cytotoxic activity in the spleen cells of mice either at
day 7 (Fig. 8 B) or day 12 (data not shown) after intracranial infection with a high dose (3 × 104 PFU) of LCMV-DOCILE. Intracranial inoculation of mice with low (30 PFU) and high (3 × 104 PFU) doses of LCMV-DOCILE
provides an example of how the extent and kinetics of virus
spread influences the kinetics of the immune response and
therefore the outcome of infection. Immunopathologic disease appears to be limited in those mice infected with a
high dose of LCMV-DOCILE because the overwhelming
antigen burden causes functional exhaustion of effector
CTLs. Functional exhaustion and subsequent deletion of
effector CTLs proceeded more slowly after intracranial infection than intravenous infection. Although this correlates
with the lower doses of LCMV that were used to infect
mice intracranially rather than intravenously, it may also reflect the delayed induction of CTLs after infection via a peripheral (intracranial; reference 35) rather than a systemic
(intravenous) route.
Production of IFN-
by GP33-specific T Cells after Intracranial Infection of B6 Mice with LCMV-DOCILE.
IFN-
has
been shown to have a direct antiviral effect after intracranial infection of mice with VV (4). In addition, exposure to
IFN-
has been shown to increase expression of MHC
class I antigens on the surface of brain cells such as astrocytes, oligodendrocytes, microglia, and neurons (36). As
shown in Fig. 9, CD8+ cells identified in the spleens (9, E
and F) and CSF (9, A and B) of mice infected intracranially
with a low dose (30 PFU) of LCMV-DOCILE, including
those that are peptide GP33 specific, exhibited a high capacity to produce IFN-
. A smaller proportion of cells recovered from both the CSF (9, C and D) and spleens (9, G
and H) of mice that had been infected with a higher dose
(3 × 104 PFU) of LCMV-DOCILE-produced IFN-
.
This correlated with the increasingly anergic status of the
cells after exposure to excessive antigen.

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Fig. 9.
Intracellular cytokine staining of
CD8+ cells isolated from the spleens and CSF
of mice infected intracranially with a low or
high dose of LCMV-DOCILE. Spleen cells
and CSF recovered from mice that had been
infected intracranially with either low (30 PFU) or high (3 × 104 PFU) dose LCMV-DOCILE were stimulated with PMA and ionomycin and subsequently stained with anti-CD8
antibodies, GP33 tetramers, and anti-IFN-
antibodies. Subsequent FACS® analysis was
carried out as described for Fig. 5. (Left) CD8+
cells that express IFN- ; (right) CD8+ GP33-specific T cells that express IFN- . The percentages of CD8+ and CD8+/tetramer+ cells
(from the experiment shown in Fig. 7) that express IFN- are shown in each panel.
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Staining of GP33-specific Cells after Intravenous Infection of
Perforin-deficient Mice with LCMV-DOCILE.
A recent study
by Sad et al. showed that GP33-specific T cell lines established by in vitro restimulation of spleen cells isolated from
LCMV-infected perforin-deficient mice (PKOB) produced more IFN-
than similar cell lines established from infected
B6 mice (37). IFN-
was measured in the supernatants of
the cultures by ELISA. In this study, spleen cells from PKOB
mice were isolated on days 9 and 15 after intravenous injection with a low dose (200 PFU) of LCMV-DOCILE.
The cells were stained with anti-CD8 antibodies, GP33
tetramers, and anti-IFN-
antibodies as described above. The results, shown in Fig. 10, A-C, indicate that the expansion of GP33-specific T cells appears to be similar at day
9 to that observed in B6 mice infected in the same way.
Both the GP33-specific cell population and the remainder
of the CD8+ cells isolated from PKOB mice did not, however, exhibit an elevated capacity to produce IFN-
when
compared to B6 mice (Fig. 11), despite the finding that virus titers were very high in PKOB mice (107 PFU LCMV/
spleen) at a time point (day 9) when virus had already been
cleared from the spleens of wild-type mice (Fig. 4). A similar analysis performed at day 15 revealed that the proportion of GP33-specific CD8+ cells had further increased in
PKOB mice (Fig. 10 E). These cells showed a reduced capacity to produce IFN-
in response to stimulation with
PMA and ionomycin (Fig. 11, compare A to C and B to
D); the mice remained persistently infected with virus (3 × 106 PFU LCMV/spleen). In a similar fashion to CD8+ cells
recovered from mice that had been infected intracranially with a high dose of LCMV-DOCILE, virus-specific CD8+
cells recovered from PKOB mice 15 d after LCMV infection also showed a reduced capacity to produce IFN-
. Virus-specific PKOB CD8+ T cells were not, however, deleted, as was observed previously in B6 mice infected
intravenously with a high dose of the same virus (Fig. 10,
compare B to C and E to F).

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Fig. 10.
Staining of GP33-specific Db-restricted CD8+ cells
isolated from the spleens of B6
and PKOB mice infected intravenously with LCMV-DOCILE.
Cells isolated from the spleens of
either B6 mice infected with a
low dose of LCMV-DOCILE
(A and D), PKOB mice infected
with a low dose of LCMV-DOCILE (B and E), or B6 mice
infected with a high dose of the
same virus (C and F) were stained
with anti-CD8 antibodies and
GP33 tetramers. (Histograms) The
gated CD8+ cell population stained
with GP33 tetramers. The results
are representative of two independent experiments carried out
using groups of two mice.
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Fig. 11.
Intracellular cytokine staining of CD8+ cells isolated from
LCMV-infected PKOB mice. Spleen cells recovered from mice that had
been infected intravenously with either low-dose (2 × 102 PFU) LCMV-DOCILE were stimulated with PMA and ionomycin and subsequently
stained with anti-CD8 antibodies, GP33 tetramers, and anti-IFN- antibodies. Subsequent FACS® analysis was carried out as described for Fig. 5.
(Left histograms) the percentage of CD8+ cells that express IFN- ; (right
histograms) the percentage of CD8+ GP33-specific T cells that express
IFN- .
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This study shows that high frequencies of CTLs are stimulated in B6 mice during the acute phase of infection with
LCMV. Frequencies of virus-specific CTLs correlate directly with the extent of early infection. Despite the induction of higher frequencies of CTLs after intravenous infection with high-dose rather than low-dose infection with
LCMV-DOCILE, these CTLs fail to control the virus and
are subsequently deleted (30). Studies on mice infected intracranially with a high dose of LCMV-DOCILE, where
the induction of virus-specific CTLs occurs in a staggered
fashion and is less rapid than after intravenous infection, indicate that an anergic phase exists between CTL induction
and deletion. During this phase, virus-specific CD8+ cells
may be described as functionally exhausted since they are characterized by a lack of cytotoxic activity and a reduced
capacity to produce IFN-
. Although the PKOB virus-specific CD8+ cells also showed a reduced capacity to produce IFN-
after prolonged exposure to antigen, these cells
were not deleted. These data indicate that disappearance of
virus-specific CD8+ T cells correlates with sustained perforin-mediated cytotoxic activity. CTL in perforin-competent mice may die as a result of interleukin starvation after
CTL-mediated destruction of LCMV-infected, cytokine-producing APCs. Alternatively, deletion of CTLs in perforin-competent mice may result directly from perforin-dependent activation-induced apoptosis. Both possibilities
remain to be evaluated.
This study further demonstrates that tetrameric class I-peptide complexes provide novel opportunities for the detection of antigen-specific T cells. The technique used in this
study differs from that described by Altman et al. (11) in
that it uses, instead of enzymatic biotinylation to the COOH
terminus of the class I heavy chain, chemical biotinylation
of the
2M subunit. This modification renders the technique versatile since the final product, biotinylated
2M,
can be used to refold any mouse or human class I heavy
chain. Use of tetrameric class I-peptide complexes has an
advantage over the use of anti-TCR antibodies in that they
allow phenotypic characterization of all T cell clones of a
given peptide-specificity. In addition, they provide the
opportunity to study the phenotype of antigen-specific T
cells without prior in vitro manipulation and without the
need for transgenic animals.
Address correspondence to Awen Gallimore, Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX39DS, UK. Phone: 44-1865-222413; Fax: 44-1865-222502; E-mail:
awen.gallimore{at}ndm.ox.ac.uk
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