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*
First Department of Internal Medicine, Research Center for Infectious Disease, Aichi Medical University, Aichi, Japan; and Departments of
Molecular and Experimental Medicine and
Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For a noncytopathic virus to persist either it must not be able to induce an effective antiviral immune response or it must be able to overwhelm or evade it. Neonatal tolerance is probably responsible for both the lack of an antiviral immune response and for viral persistence following mother-infant transmission, which is the most common antecedent of persistent HBV infection worldwide (1). The immunologic basis for viral persistence during adult-onset infection, however, is not well understood. In view of the repeated observation that the CTL response to HBV is much less vigorous in chronically infected patients than it is during acute infection (5, 6, 7, 8), reasonable candidates are the induction of peripheral tolerance or exhaustion of the T cell response, inhibition of Ag presentation, selective immune suppression, down-regulation of viral gene expression, and viral mutations that abrogate, anergize, or antagonize Ag recognition by virus specific T cells (reviewed in 1 . While all of these alternatives are possible, direct evidence exists only for the escape mutation scenario (9).
We have argued against this hypothesis as a cause of HBV persistence because many conditions must be fulfilled that rarely occur during HBV infection for a mutant virus to be selected by CTL-mediated immune pressure (reviewed in 1 . Perhaps the most important condition is the occurrence of a strong CTL response that is focused on a single viral epitope. This type of CTL response is unusual both during acute HBV infection, when the CTL response is typically directed against multiple epitopes, and during chronic hepatitis, when it is usually weak or undetectable (6, 7, 10, 11). Accordingly, we have found selection of CTL escape variants to be very uncommon during chronic HBV infection (12). Nonetheless, strong and narrowly focused CTL responses are seen occasionally in these patients, and in this setting viral escape mutations can occur (9). Vigorous oligoclonal expansions of T cells have also been described in other persistent viral infections, especially HIV (13, 14, 15, 16). Even in these infections, however, viral mutations that affect recognition of an epitope by some CTL clones do not automatically affect all CTL clones specific for the same epitope, since different T cell clones can recognize different residues in the same epitope (17, 18).
The current study was performed to determine the extent to which epitope-inactivating mutations can lead to escape from the CTL response by defining the molecular anatomy of an immunodominant CTL epitope using a large panel of independently derived CTL clones. The data indicate that the CTL response to individual viral epitopes can be extremely diverse, supporting the idea that selection of TCR contact site escape variants is probably not responsible for the establishment of viral persistence during HBV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P815 cells (H-2d) were transfected with pMAMneo vector (Clontech, Palo Alto, CA) into which the HBV large envelope open reading frame (ayw subtype) had been introduced (19) to generate a cell line designated P815-preS1, and with an EBO vector (20) into which the HBV small open reading frame (ayw subtype) had been introduced, designated P815-S. Cell lines Dnorm, Knorm, and W12.1 were produced by introducing the Dd, Kd, and Ld genes, respectively, into the KOL fibroblastoma cell line (H-2k) and were provided by Dr. James Forman (Southwestern Medical Center, Dallas, TX) (21, 22). RMA-S Ld cells are stable transformant of RMA-S cells and express the Ld molecule; they were provided by Dr. Herman N. Eisen (Massachusetts Institute of Technology, Cambridge, MA). All the cell lines except for RMA-S Ld were grown in DMEM (Life Technologies, Grand Island, NY) containing 10% FCS, L-glutamine (2 mM), streptomycin (50 µg/ml), penicillin (50 U/ml), HEPES buffer (15 mM), and anti-PPLO (HyClone, Logan, UT; 6 mg/ml). RMA-S Ld cells were maintained in RPMI 1640 (Life Technologies) supplemented as described above. Recombinant vaccinia viruses expressing the HBV large (HBVenv-vac) and major (HBs-vac) envelope polypeptides (ayw subtype) were derived as previously described (7).
Synthetic peptides
Peptides were synthesized by Chiron Technologies (Clayton, Australia), Multiple Peptide Systems (San Diego, CA), and the Peptide Synthesis Core Facility at The Scripps Research Institute (La Jolla, CA). All peptides were dissolved in 100% DMSO (Mallinckrodt, Paris, KY) at a concentration of 5 or 10 mg/ml and diluted to 1 mM with DMEM, after which they were stored at -20°C until use.
CTL induction
Murine HBsAg-specific CTLs were induced as previously described (22, 23). B10.D2 mice (H-2d) were immunized two or three times i.p. at 7-day intervals with 1 x 108 plaque-forming units of HBs-vac. Spleen cells were harvested 7 days after the last injection, and splenocytes (4 x 106 cells/well) were cultured with irradiated (20,000 rad) P815-env transfectants (1 x 105 cells/well; P815-preS1 or P815-S) or with 20 µg/ml of an antigenic peptide (HBs21–40), previously shown to contain an immunodominant Ld restricted CTL epitope (19), in complete Eagle Hank’s amino acid (EHAA) culture medium (Life Technologies) containing streptomycin (50 µg/ml), penicillin (50 U/ml), anti-PPLO (HyClone, Logan, UT; 6 mg/ml), 10% FCS, 2-ME (5 x 10-5 M), and 2.5% EL-4 supernatant, as source of T cell growth factor, in 24-well plates (Corning, Corning, NY). The primed spleen cells were restimulated two or more times at 7-day intervals, and 7 days after the last stimulation they were cloned in 96-well round-bottom plates (Corning) in EHAA medium at 0.3–1 cell/well with irradiated (20,000 rad) P815-preS1 (1 x 104/well) and irradiated (2,000 rad) syngeneic splenocytes (4 x 105/well). Wells containing growing cells were expanded and tested for HBsAg-specific cytotoxic activity as described below.
CTL assay
The cytolytic activity of T cell lines and clones was assessed
in a standard 4-h cytotoxic assay with 51Cr-labeled target
cells as previously described (22, 23). The P815 cells and derivative
transfectants were labeled directly from culture. The KOL fibroblastoma
cells and derivative transfectants were incubated with 50 U/ml rIFN-
for 24 h before analysis to insure MHC class I expression. All
experiments were performed in duplicate. The percent specific
51Cr release was derived as follows: (experimental cpm
- spontaneous cpm/total cpm - spontaneous cpm) x 100.
Spontaneous release was always <20% of the total.
Competition assay
After labeling with 51Cr and extensive washing, target cells (P815) were cultured in complete DMEM and plated (5 x 103/cells) in 96-well flat-bottom plates. They were incubated with various concentrations of the variant peptides for 1 h, followed by a suboptimal concentration (0.001 µM) of wild-type peptide for another hour before addition of the effector CTL clones.
Antagonism assay
After labeling with 51Cr and extensive washing, target cells (P815) were cultured, plated (5 x 103/cells, 96-well flat-bottom plates), and incubated with a suboptimal concentration (0.001 µM) of wild-type peptide for 1 h. After washing, target cells were incubated with varying concentrations (up to 5 µM) of the variant peptides for another hour before addition of the CTL clones.
TCR ß-chain analysis
TCR BV gene usage of CTL clones was first determined by a BV multiprobe RNase protection assay, and then the appropriate BV chains were obtained by RT-PCR. The details of the RNase protection assay encompassing 18 BV genes have been previously described (24). Clonal BV gene expansions were readily apparent without quantitation. RT-PCRs were performed using cDNA from 20 ng of total cellular RNA, specific sense BV and antisense BC primers (25) at 2 ng/µl, and standard reagents (Boehringer Mannheim, Indianapolis, IN). PCR conditions were 30 cycles of 94°C for 30 s, 48–50°C for 30 s depending on the primer, and 72°C for 40 s. PCR products were either sequenced directly by fluorescent cycle sequencing (Perkin-Elmer Applied Biosystems, Foster City, CA) or cloned into the pCRII plasmid (Invitrogen, Carlsbad, CA) and then sequenced (Amersham, Arlington Heights, IL).
Cytokine production
For measurements of TNF-
, IFN-, IL-2, IL-4, and IL-10
production, 1 x 106 cloned CTL were grown in 24-well
plates in the presence of 1 x 106 P815-preS1
stimulator cells in complete EHAA medium without EL-4 supernatant.
Forty-eight hours after the stimulation, the culture supernatant was
collected and stored at -20°C until ELISA analysis using
commercially available reagents (Genzyme, Cambridge, MA) according to
the manufacturer’s instruction.
Ld binding assay
The RMA-S peptide binding assay was performed as described previously (26, 27). In brief, RMA-S Ld cells were cultured overnight at 25°C to increase the cell surface expression of empty class I molecules. Cells (1 x 105/well) were plated (96-well plates) in 100 µl of RPMI containing 2% FCS plus peptide (final concentration, 0.3 µM). After overnight incubation at 25°C, the cells were washed and suspended in HBSS supplemented with 2% horse serum. mAb against Ld (30-5-7S) was added, and incubation took place on ice for 30 min followed by washing. Subsequently, the cells were incubated with FITC-labeled goat anti-mouse F(ab')2 for 30 min on ice, after which they were washed and analyzed in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Ten thousand cells were gated and analyzed for fluorescence intensity. The fluorescence index was calculated as follows: (MFI in the presence of peptide - MFI in the absence of peptide)/(MFI in the absence of peptide). The mean fluorescence intensity (MFI) is the mean channel number of 1 x 104 gated cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eleven HBsAg-specific CTL clones established from three immunized
B10.D2 mice were used for this study. The immunization and in vitro
expansion protocols used before cloning by limiting dilution are shown
in Table I
. Clones that displayed good
growth characteristics and the ability to specifically lyse >30% of
the P815-preS1 target cells at an E:T cell ratio of 5 were selected for
this study. All CTL clones recognized P815-preS1 target cells with
approximately the same efficiency (Fig. 1A). As displayed for clone
6C2 in Fig. 1B, we showed that all the CTL clones displayed
the same fine specificity, using target cells pulsed with a panel of
truncated synthetic peptides corresponding to an immunodominant CTL
epitope located between residues 28–39 of HBsAg (HBs28–39) that we
have previously described (22). Next, using peptide pulsed (HBs28–39,
1 µM) H-2k-positive mouse fibroblasts that express
Kd, Dd, or Ld as target cells, we
showed that all the CTL clones recognized the epitope in the context of
the Ld molecule (Fig. 1C). Peptide dose
titration experiments demonstrated that the CTL clones displayed
comparable epitope binding affinity (Fig. 1D). Importantly,
as shown in Table II, all the clones
produced comparable amounts of IFN- and TNF- following Ag
stimulation. Finally, as shown in Fig. 2,
TCR ß-chain analysis revealed six distinct clonal populations
among the 11 CTL clones studied, three derived from mouse 1, two
derived from mouse 2, and one derived from mouse 3, with no specific
ß-chain preference. Not surprisingly, CTLs were identified that used
the same TCR BV chain but different BD and BJ segments. Importantly,
none of the populations dominated the others. Collectively, these
results indicate that CTL clones that display widely varying TCR
ß-chain usage can recognize the same CTL epitope with similar
affinity in the context of the same MHC restriction element, and that
their functional responses are comparable upon recognition of this
epitope. The results also demonstrate that the immunization and in
vitro stimulation protocols used to produce the CTL clones can
influence the response at the level of TCR repertoire diversity.
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A set of 48 peptides containing alanine (A), tyrosine (Y),
glutamic acid (E), and arginine (R) substitutions at each position
(Table III) was used to define the MHC
and TCR contact sites in the wild-type epitope (IPQSLDSWWTSL) and to
analyze the fine specificity of the 11 CTL clones. The binding affinity
of these peptides to Ld was examined with an Ld
-transfected, TAP-deficient RMA-S cell line by FACS quantitation of
cell surface Ld expression after addition of the peptides.
As shown in Fig. 3 (lower
panel), all four substitutions at positions 2 and 12 strongly
reduced the Ld binding affinity of the peptides, implying
that these are dominant anchor residues for this epitope. Other
positions (especially positions 5 and 10) also contribute to
Ld binding, but to a lesser degree, since Ld
expression was less profoundly reduced by substitutions at these
positions and some of the substitutions at these positions did not
reduce binding. In parallel experiments, we analyzed the ability of
each of the 11 CTL clones to kill 51Cr-labeled target cells
that had been pulsed with a series of concentrations of the wild-type
peptide and each of the 48 substituted peptides. As shown in the
upper panel of Fig. 3 for clone 6C2 at an E:T cell ratio of
10 and a peptide concentration of 0.1 µM, substitutions at several
positions, including the presumptive anchor positions 2 and 12, reduced
or abolished CTL recognition. Similar experiments were performed for
each clone. In each instance, several substitutions were identified
that inhibited CTL recognition without affecting Ld binding
affinity (e.g., positions 1, 4, 6, and 7 in the case of clone 6C2),
suggesting that the corresponding wild-type residues probably serve as
TCR contact sites for that clone. Peptide competition experiments were
performed to test this hypothesis.
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Once the substitutions that abrogated peptide recognition were
identified for each CTL clone, the corresponding substituted peptides
were incorporated into a competition assay to monitor their ability to
block the binding of the wild-type peptide to the Ld
molecule. Briefly, 51Cr-labeled P815 target cells were
preincubated with the substituted peptides at several concentrations
that did not sensitize the target cells to killing by the CTL clone.
The wild-type peptide was then added at a concentration (in most cases
0.001 µM) that had been previously determined to sensitize the target
cells to approximately half-maximal lysis by the corresponding CTL
clone. After 1 h, the CTL clones were added and a 4-h
51Cr release assay was performed. If the substituted
peptide binds to Ld and if it is used in sufficient (at
least 100-fold) molar excess, it will saturate the available
Ld binding sites and block binding by the wild-type peptide
such that the percent specific 51Cr release induced by the
corresponding CTL clone will be reduced. On the contrary, if the
substituted peptide does not bind to Ld, it will not
inhibit binding by the wild-type peptide, and the percent specific
51Cr release will not be reduced. Sample results for clone
6C2 and two substitutions, i.e., Pro
Tyr at position 2 and SerAla
at position 4, are shown in Fig. 4. In
the position 2 substitution (Fig. 4, upper panel),
increasing concentrations of the substituted peptide did not reduce
recognition of the wild-type peptide, suggesting that the ProTyr
substitution at position 2 abrogated Ld binding. This is
compatible with the Ld binding assay results shown in Fig. 3
(lower panel), indicating that position 2 is an MHC
contact site. In the position 4 substitution (Fig. 4, lower
panel), increasing concentrations of the substituted peptide
inhibited recognition of the wild-type peptide, suggesting that
the SerAla substitution at position 4 did not reduce the
Ld binding capacity ability of the peptide and indicating
that this position is a TCR contact site for clone 6C2.
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. In every instance, positions 2 and 12
were shown to be MHC binding sites, confirming the results of the
binding assay (Fig. 3, lower panel). In contrast, several
different positions were served as TCR contact sites for the various
clones, the specificity of which was clearly determined at the level of
the TCR, since each ß-chain displayed its own unique specificity
pattern and because there were no discrepancies between clones that
used the same ß-chain. For example, threonine at position 10 was
uniquely recognized as a TCR contact site by 2 clones (13F6 and 14F7)
that share the same ß-chain, which is not used by any of the other
clones. Other positions were recognized by multiple TCRs. For
example, isoleucine at position 1 was recognized by five clones
representing two different ß-chains, serine at position 4 was
recognized by nine clones representing four different ß-chains,
etc. Importantly, aspartate at position 6 was the only the TCR contact
site that was recognized by all the clones. Interestingly, as shown in
Fig. 5, leucine and threonine at positions 5 and 10, respectively,
appear to be able to function as both MHC binding sites and TCR contact
sites depending on the clone.
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To further examine the diversity of the TCR repertoire, a set of
peptides containing every possible substitution at positions 4 and 6
(Fig. 6) was tested for recognition by
three separate clones, two of which (6C2 and 11B5) used the same
ß-chain. In a preliminary experiment we established that the
Ld binding affinity of these peptides was comparable to
that of the wild-type peptide (data not shown). As shown in Fig. 7, hydrophobic and charged substitutions
at both positions were generally incompatible with recognition by
clones 6C2 and 11B5. In contrast, many of these substitutions were
recognized by clone 13F6, while certain polar substitutions
(e.g., Y and H) that were tolerated by clones 6C2 and 11B5 abrogated
recognition by clone 13F6, reflecting its different TCR.
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There are several reports that substitutions of TCR contact sites
of the antigenic peptide can yield analogue peptides that can still
interact with the TCR but be unable to deliver a full stimulatory
signal, thus acting as an antagonist for the TCR (28, 29, 30, 31, 32, 33, 34). To examine
whether the substitutions at the dominant TCR contact sites (positions
4 and 6) may result in variant sequences with an antagonistic effect on
TCR recognition of the wild-type peptide (HBs28–39), antagonism assays
were performed with clone 6C2 using the same set of peptides containing
all possible substitutions at positions 4 and 6 (Fig. 6). Briefly,
51Cr-labeled P815 target cells were preincubated with the
wild-type peptides at a suboptimal concentration (0.001 µM) and
washed, and the substituted peptides (up to 5 µM) were added and
incubated for 1 h, after which the CTL clone was added and a 4-h
51Cr release assay was performed. None of the substituted
peptides blocked the ability of clone 6C2 to kill target cells pulsed
with up to 5 µM of the wild-type peptide (not shown), indicating that
none of the position 4 or 6 substitutions created an antagonistic
variant of this epitope with respect to clone 6C2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonetheless, the role of CTL escape in viral persistence remains
controversial. The current study was undertaken, therefore, to examine
the requirements for an epitope to escape recognition by the CTL
response, since the same requirements should apply to all the epitopes
normally recognized during a viral infection. To simplify the analysis,
a single strain of inbred mice (B10.D2) was primed to produce a CTL
response to a single viral protein, HBsAg. We and others have
previously demonstrated (22, 47) that the CTL response to this protein
in these mice is primarily directed against a single, unusually long
(12-mer), immunodominant, Ld-restricted epitope located
between residues 28–39 of the major envelope protein of HBV
(IPQSLDSWWTSL). As summarized in Fig. 5, during this study we
demonstrated that positions 2 and 12 are dominant anchor residues
responsible for the ability of the Ld molecule to bind the
epitope, and that positions 5 and 10 can function as minor anchor
residues. We also demonstrated that residue 1 and 4–10 can serve as
TCR contact sites depending on the CTL clone being studied.
Importantly, we showed that the CTL response to this epitope is
remarkably polyclonal (involving several different TCR ß-chains) and
multispecific (each ß-chain displaying a unique fine specificity
profile), emphasizing the impressive diversity of the CTL response at
the level of a single epitope, as previously reported (48, 49, 50, 51, 52).
As shown in Fig. 7, several distinct chemical interactions account for
TCR recognition of the MHC-peptide complex. For example, in the case of
position 4, amino acid polarity is important for TCR recognition by
clones 6C2 and 11B5 (S to polar group amino acid substitution is
acceptable for TCR recognition), while in the case of position 6, the
size or the shape of the side chain seems to be important (D to N
substitution is most acceptable). Recognition of both of these
positions by clone 13F6, however, does not follow these rules,
suggesting that the shape of the exposed peptide surface or the
framework of the peptide binding groove is recognized by this clone’s
TCR. These observations and other results reported herein are pertinent
to the question of viral escape from the CTL response. For example,
Fig. 5 demonstrates that nonconservative substitutions at nine of the
12 positions in this epitope either had no effect on recognition by any
of the CTL clones, or they affected recognition by some of the clones
but not others. Thus, mutations involving 75% of the epitope would not
be expected to abolish recognition by all the CTL clones. Furthermore,
although certain substitutions at positions 2, 6, and 12 abrogated
recognition of the epitope by all the CTL clones, many substitutions
either did not or should not affect recognition by any of them. Since
the Ld binding motif includes P and Q at position 2 and
hydrophobic residues at the C-terminus (in this case position 12), the
corresponding substitutions at these positions should be tolerated.
Likewise, examination of Fig. 7 reveals that only seven of the possible
19 substitutions at TCR contact position 6 abrogated recognition by the
three CTL clones tested, and only six substitutions at TCR contact
position 4 (which is not recognized by all of the clones) had this
effect. It is important to note that even this number of tolerable
substitutions at these TCR contact sites is probably an overestimate,
since only three CTL clones were studied in this manner and also
because the CTL clones studied certainly do not represent the entire
TCR repertoire for this epitope. Finally, the considerations described
herein for epitope-inactivating mutations also apply to mutations that
generate TCR antagonists (not observed in this study). Specifically,
because of the TCR diversity of the CTL response to any given epitope,
mutations that function as antagonists for one CTL clone need not, and
usually do not, have the same effect on others.
Interestingly, the competition studies summarized in Fig. 5 indicate
that leucine and threonine at positions 5 and 10, respectively, can
function as TCR contact sites for certain clones, although the
Ld binding assay clearly demonstrated that they also
function as minor MHC binding sites for this epitope (see Fig. 3, lower panel). Perhaps the simplest explanation for this
seeming paradox is that because of its unusual length, this epitope can
display more than one stable orientation in the Ld binding
groove, one of which causes these residues to bind Ld,
while they are free to interact with the TCR in the other orientation.
According to this scenario, the position(s) would score as a TCR
contact site for CTL clones that require interaction with this
residue(s) for activation. Alternatively, the position(s) would score
as an MHC binding site(s) for clones that do not recognize this
residue(s) as long as the substitution reduced the amount of target
cell-associated peptide below the threshold needed for recognition by
the CTL clone being tested. If not, the position would score as
neutral, as shown for some of the clones in Fig. 5. Resolution of this
interesting paradox will require structural analysis of the peptide-MHC
complex.
In conclusion, the current results demonstrate that the CTL response to
a single viral epitope can be extremely diverse. Since all the CTL
clones displayed comparable cytolytic activity (Fig. 1) and cytokine
expression profiles (Table II), and since no single TCR was dominant
(Figs. 2 and 5), most of the mutations that might occur in this epitope
are not likely to abolish all CTL effector activity. In the rare
instance in which a mutation leads to escape from all CTL clones of a
single specificity, the concomitant CTL response to other viral
epitopes, each of which could be as diverse as the response described
in this report, make it very unlikely that mutational inactivation of a
single CTL epitope will lead to viral persistence in our opinion. Some
have argued that the CTL response can be narrowly focused early during
viral infections (41, 53). If this is correct, CTL escape mutations
could contribute to viral persistence by retarding the rate of viral
clearance. However, the diversity of the CTL response to individual
epitopes illustrated herein suggests that even under these conditions,
escape from all CTL clonal specificities would not be guaranteed.
Furthermore, the procedures used to monitor the CTL response during
infection should be as unbiased as possible to avoid artificially
restricting the CTL repertoire and inappropriately viewing the response
as narrow when, in fact, it might be quite diverse.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francis V. Chisari, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; HBs21–40, residues 21–40 of hepatitis B surface antigen; EHAA, Eagle Hank’s amino acid; MFI, mean fluorescence intensity.
Received for publication May 4, 1998. Accepted for publication July 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain to cross-reactive CD+8 CTL clone. J. Immunol. 153:4070.[Abstract]
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R. Schirmbeck, X. Zheng, M. Roggendorf, M. Geissler, F. V. Chisari, J. Reimann, and M. Lu Targeting Murine Immune Responses to Selected T Cell- or Antibody-Defined Determinants of the Hepatitis B Surface Antigen by Plasmid DNA Vaccines Encoding Chimeric Antigen J. Immunol., January 15, 2001; 166(2): 1405 - 1413. [Abstract] [Full Text] [PDF]
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D.-G. Lim, K. Bieganowska Bourcier, G. J. Freeman, and D. A. Hafler Examination of CD8+ T Cell Function in Humans Using MHC Class I Tetramers: Similar Cytotoxicity but Variable Proliferation and Cytokine Production Among Different Clonal CD8+ T Cells Specific to a Single Viral Epitope J. Immunol., December 1, 2000; 165(11): 6214 - 6220. [Abstract] [Full Text] [PDF]
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V. Pasquetto, S. Wieland, and F. V. Chisari Intracellular Hepatitis B Virus Nucleocapsids Survive Cytotoxic T-Lymphocyte-Induced Apoptosis J. Virol., October 15, 2000; 74(20): 9792 - 9796. [Abstract] [Full Text]
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I. E. A. Flesch, D. Stober, R. Schirmbeck, and J. Reimann Monocyte inflammatory protein-1{alpha} facilitates priming of CD8+ T cell responses to exogenous viral antigen Int. Immunol., September 1, 2000; 12(9): 1365 - 1370. [Abstract] [Full Text] [PDF]
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J. Wild, M. J. Grusby, R. Schirmbeck, and J. Reimann Priming MHC-I-Restricted Cytotoxic T Lymphocyte Responses to Exogenous Hepatitis B Surface Antigen Is CD4+ T Cell Dependent J. Immunol., August 15, 1999; 163(4): 1880 - 1887. [Abstract] [Full Text] [PDF]
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