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Fas-Mediated Apoptosis Causes Elimination
of Virus-Specific Cytotoxic T Cells in the
Virus-Infected Liver
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of June 14, 2017.
Zhang-Xu Liu, Sugantha Govindarajan, Shigefumi Okamoto
and Gunther Dennert
J Immunol 2001; 166:3035-3041; ;
doi: 10.4049/jimmunol.166.5.3035
http://www.jimmunol.org/content/166/5/3035
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References
Fas-Mediated Apoptosis Causes Elimination of Virus-Specific
Cytotoxic T Cells in the Virus-Infected Liver1
Zhang-Xu Liu, Sugantha Govindarajan, Shigefumi Okamoto, and Gunther Dennert2
Immunity to allogeneic MHC Ags is weak in rodent livers, raising questions as to the mechanisms that might control responses
in this organ. Infection with an adenovirus vector reveals that T cell-mediated immunity to nonself-Ags in the liver is self-limiting.
Virus-induced liver injury decreases and coincides with disappearance of virus-specific CTL, concomitant to an increase of
apoptotic T cells early after infection. But whereas death in CD4 cells is independent of Fas, perforin, and TNF-␣, that of CD8
cells requires Fas and not perforin or TNF-␣ pathways. Fas ligand is expressed on liver-infiltrating cells, pointing to death by
fratricide that causes almost complete disappearance of virus-specific CTL 4 wk after infection. CTL elimination is virus dose
dependent, and high doses induced high alanine aminotransferase values, elevated expression of Fas ligand on CD8 cells, and
increased CD8 cell migration into the infected liver. The Journal of Immunology, 2001, 166: 3035–3041.
Department of Molecular Microbiology and Immunology, University of Southern
California/Norris Comprehensive Cancer Center, Keck School of Medicine at University of Southern California, Los Angeles, CA 90089
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Received for publication August 1, 2000. Accepted for publication December 18,
2000.
1
This work was supported by Public Health Service Grants CA 59318, AI 43954, and
AI 40038.
2
Address correspondence and reprint requests to Dr. Gunther Dennert, University of
Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue,
M/S #73, Los Angeles, CA 90089-9176. E-mail address: [email protected]
3
Abbreviations used in this paper: HBV, hepatitis B virus; HBC, hepatitis C virus;
B6, C57BL/6; FasL, Fas ligand; PKO, B6-pfptm1Sdz; lpr, B6.MRL-Faslpr; gld,
B6Smn.C3H-FasLgld; ALT, alanine aminotransferase; ELISPOT, enzyme-linked immunospot assay; lacZ, ␤-galactosidase.
Copyright © 2001 by The American Association of Immunologists
fections. In fact, a number of viruses, among them hepatitis B
(HBV)3 and hepatitis C virus (HCV), are known to cause chronic
infections, often leading to liver cirrhosis and death (5–7). Therefore, the question arises whether the failure of the liver to successfully eliminate viral infections may in part be because of its tolerance-inducing activity. To investigate this, use was made of a
viral infection model in which a replication-deficient adenovirus,
expressing the ␤-galactosidase (lacZ) gene, is injected into mice.
This had been shown to result in massive infection and viral gene
expression in the liver, which in turn induces a strong T cellmediated immune response (8, 9). Here we report that the T cell
response to viral Ags, expressed in the liver, is terminated at early
times after the infection by the induction of cell death in both CD4
and CD8 cells. Induction of cell death in CD8 cells is shown to
involve Fas, leading to elimination of CTLs able to respond to
virus-infected cells as early as 6 days after the infection. It is concluded that the liver induces unresponsiveness to non-MHC Ags
by a clonal deletion mechanism.
Materials and Methods
Animals, immunization, and assay for serum alanine
aminotransferase (ALT)
Pathogen-free female C57BL/6 (H-2b) (B6), B6-pfptm1Sdz (PKO),
B6.MRL-Faslpr (lpr), and B6Smn.C3H-FasLgld (gld) mice, 6 –12 wk of age,
were obtained from The Jackson Laboratory (Bar Harbor, ME). B6/
TNFR1/R2 double-deficient (TNFR1/R2KO) mice were generated and
generously provided by Dr. Chaim Jacob (Department of Medicine, University of Southern California, Keck School of Medicine, Los Angeles,
CA). Bone marrow chimeras were prepared by irradiating mice 950 rad,
followed by i.v. injection of 2 ⫻ 107 bone marrow cells and 5 ⫻ 107 spleen
cells. Mice were used for virus infection 1 wk later. For immunization, type
5 adenovirus with deletions in the E1 and E3 region and carrying the lac
Z gene was purchased from Microbix Biosystems (Toronto, Ontario, Canada). Virus was propagated in 293 cells as recommended by the supplier.
Animals were primed by injection of virus into the tail vein on day 0 (10,
11). Serum ALT was assayed as described (10), with a commercial assay
kit (Sigma, St. Louis, MO). For IL-2 treatment, mice were injected with
4 ⫻ 105 U recombinant mouse IL-2 (BD PharMingen, San Diego, CA) per
day i.p., starting with the day of virus infection until completion of the
experiment.
Preparation of liver lymphocytes and fluorometric analysis
Mononuclear cells were isolated from livers as described previously (10, 11).
Liver tissue was passed through a 200-gauge stainless steel mesh in serum-free
HBSS, and mononuclear cells were purified by Percoll gradient centrifugation.
RBC contained in the mononuclear cell preparation were lysed by ammonium
0022-1767/01/$02.00
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T
he mechanisms regulating immune responses to nonselfAgs in the liver have remained mysterious despite much
experimental effort to understand them (1). Unexplained
is the finding that liver transplants between MHC-disparate mouse
strains survive indefinitely without the need for immunosuppression (2). This observation raises fundamental questions as to why
this organ may escape T cell-mediated allorejection mechanisms in
face of rapid destruction of allogeneic skin, heart, and kidney
grafts. A possible clue as to the underlying mechanisms is provided by the demonstration that mice who have accepted allogeneic livers express tolerance to skin grafts of liver donor origin (3).
Therefore, the murine liver is able to induce unresponsiveness to
MHC Ags. The principal mechanisms inducing unresponsiveness
have not been elucidated but appear to involve induction of T cell
anergy as spleen cells from transplant recipients retain the ability
to respond in vitro to MHC Ags of the transplant (4). Therefore, an
important question is how is the liver able to induce anergy in T
cells and is induction of anergy the only mechanism by which
unresponsiveness to the transplant is maintained. Moreover, the
induction of anergy may not be the principal mechanism by which
unresponsiveness to other than allo-MHC Ags may be induced in
the liver.
Understanding the immunoregulatory pathways by which the
liver induces unresponsiveness is of much interest, not only to
facilitate successful liver transplantation but also to overcome failure of the immune system to successfully eliminate microbial in-
3036
Fas-MEDIATED ELIMINATION OF VIRUS-SPECIFIC CTL IN LIVER
chloride. For FACS analysis, cells were stained with mAbs as described (11).
The following Abs were used: anti-mouse Fc␥ receptor CD16/CD32 (2.4G2),
biotin anti-mouse Fas ligand (FasL; MFL3), and PE- or FITC-conjugated antiCD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), and anti-NK1.1
(PK136), purchased from BD PharMingen. For annexin V staining, cells were
first incubated with PE-conjugated anti-CD3, anti-CD4, and anti-CD8 followed by staining with the FITC-annexin V detection kit, according to the
manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis,
IN). The percentage of annexin V-positive cells in the CD3⫹, CD4⫹, and
CD8⫹ cell populations were calculated. For FasL staining, cells were incubated with anti-mouse Fc␥ receptor mAb CD16/CD32 at 4°C for 10 min, then
stained with biotin-conjugated anti-FasL at 4°C for 30 min. After washing,
cells were stained at 4°C for 30 min with PE- or FITC-conjugated streptavidin
and FITC- or PE-conjugated mAb against CD3, CD4, CD8, and NK1.1. FACS
analysis was performed on a FACStarPlus (Becton Dickinson, Mountain View,
CA). The number of CD3⫹, CD4⫹, and CD8⫹ cells per liver was calculated
by multiplying the measured percentage of each subpopulation with the total
number of mononuclear cells per liver.
Induction of in vitro CTL responses and enzyme-linked
immunospot (ELISPOT) assays
RT-PCR assays
Liver tissue was harvested and total RNA extracted by the phenol/chloroform method by using RNAzol B kit (Tel-Test, Friendswood, TX) (10, 11).
RNA (5 ␮g) was reverse-transcribed to cDNA in a 50-␮l reaction mixture
with Superscript II RNase H⫺ reverse transcriptase and random primers
(Life Technologies, Rockville, MD). For PCR, the equivalent amount of
cDNA product (5 ␮l) was amplified in a 50-␮l reaction mixture containing
10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U
Taq DNA polymerase (Perkin-Elmer, Norwalk, CT), and 1 ␮M of each
specific primer. The amplification was performed in a Thermoline Gene E
thermocycler (Techne, Cambridge, U.K.) set at 1 min each at 94°C, 58°C,
and 72°C for 35 cycles for perforin, FasL, and TNF-␣ and 30 cycles for
␤-actin, followed by an extension at 72°C for 7 min. After amplification,
PCR products were electrophoresed on a 2% agarose gel and visualized by
ethidium bromide staining under UV illumination. The specific primers for
␤-actin and TNF-␣ were obtained from Stratagene (La Jolla, CA). Primers
for perforin and FasL were: perforin, sense 5⬘-CAC AAGTTC GTG CCA
GGT GTA-3⬘, antisense 5⬘-GCA TGC TCT GTG GAG CTG TTA-3⬘ (13);
FasL, sense 5⬘-CTG GAA TGG GAA GAC ACA TA-3⬘, antisense 5⬘AAA GGT CTT AGA TTC CTC AA-3⬘ (14).
Histological procedures
To stain lacZ, livers were harvested and immediately frozen in liquid nitrogen. Frozen sections (6 ␮m) were fixed with 0.5% glutaraldehyde in
PBS for 10 min, rinsed twice for 10 min with PBS containing 1 mM
MgCl2, and incubated with X-Gal solution (1 mg/ml X-Gal, 5 mM
K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mM MgCl2 in PBS) at 37°C, 100%
humidity for 4 h, then washed twice with distilled water (10, 11).
Results
CTLs able to respond to adenovirus Ags disappear after virus
infection
Injection of adenovirus constructs coding for the lacZ gene (adeno
lacZ) into the tail vein of mice results in infection and viral gene
expression in the liver (8, 9). This in turn triggers an immune
response that is reflected in mononuclear cell infiltration and increase of liver enzymes in the serum (10, 11). To monitor viral
gene expression, liver sections were stained for lacZ activity, and
the percentage of positive cells was determined microscopically.
Fig. 1 shows that the percentage of lacZ-expressing hepatocytes is
stable during the first 15 days but thereafter decreases and becomes
undetectable 4 wk after the infection. To correlate viral gene expression with liver injury, sera were assayed for the presence of
liver enzymes. As shown in Fig. 1, ALT values increase by day 3,
peak on day 9, and have decreased by day 15, i.e., at a time at
which no decrease in the expression of lacZ is noticeable. This
early decrease of ALT levels, i.e., before a decrease of lacZ-expressing hepatocytes, raises the question as to the reason for the
premature termination of liver injury.
The principal effector cell, responsible for viral gene elimination
in this model has been shown to be the CTL (8, 9). Therefore, it is
important to assess the activity of virus-specific CTL during the
course of infection. Spleens were harvested from infected mice and
tested in vitro for an allogeneic and virus-specific CTL response.
Fig. 2A shows that cultures from spleens harvested before the infection or on days 10 and 28 thereafter respond equally well with
the induction of CTL to H-2d stimulator cells. In contrast, responses to virus Ags show a much different pattern. There is a low
but detectable response in unprimed spleens and a high response in
cells from mice harvested 10 days after the infection (Fig. 2B). In
contrast, spleen cells from day 28 show a response that is not
higher than that of unprimed controls (Fig. 2B). These results show
that while priming of virus specific CTL is demonstrable at an
early time after the infection, it is undetectable 4 wk later.
T cell apoptosis occurs in the liver shortly after the viral
infection
The disappearance of primed CTL from the spleen of virus-infected mice raises the question of mechanisms that might be involved. Primed CTL could have left the spleen, or they could have
been anergized or been eliminated by induction of cell death. To
examine the latter possibility, liver mononuclear cells were isolated from infected mice, stained for annexin V, and analyzed by
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Spleen cells were stimulated with irradiated allogeneic stimulator cells (H2d) or 5 PFU virus per input cell in 2-ml Linbro plates (Becton Dickinson,
Franklin Lakes, NJ) for 5 days as described (11). Target cells for cytotoxicity assay were P815 (H-2d) or C57SV (H-2b) infected with 50 PFU of
virus per cell then incubated for 24 h at 37°C (11). To demonstrate that cell
lysis is attributable to CTL, effector cells were treated before assay with
anti-CD8 Ab (11) and Low-Tox complement (Accurate Chemical and Scientific, San Diego, CA). For IFN-␥ ELISPOT assays (11, 12), spleen cells
were restimulated in vitro with virus for 5 days and then seeded with
virus-infected C57SV cells in 96-well plates. To prepare plates, 100 ␮l of
10 ␮g/ml purified anti-IFN-␥ (R4-6A2; BD PharMingen) was pipetted into
Multiscreen 96-well filtration plates (Millipore, Bedford, MA) and incubated overnight at 4°C. Plates were washed three times and a suspension of
1 ⫻ 106 C57SV cells and 1 ⫻ 105 spleen cells in 200 ␮l RPMI 1640 10%
FCS was added per well and incubated for 24 h at 37°C. After washing the
plates three times in PBS 0.05% Tween 20, 100 ␮l of a solution containing
0.05% Tween 20, 1% BSA, and 5 ␮g/ml biotin conjugated anti-IFN-␥ Ab
(XMG1.2; PharMingen) in PBS was pipetted into each well and plates
incubated overnight at 4°C. A total of 100 ␮l of a solution containing a
1/400 dilution of 1 mg/ml avidin peroxidase (Sigma) in PBS containing
0.05% Tween 20 and 1% BSA was added per well. After 2 h at room
temperature, plates were washed four times in PBS 0.05% Tween 20. Into
each well, 200 ␮l of ABE solution (Zymed, South San Francisco, CA) was
pipetted, followed by incubation for 15 min in the dark. After washing in
double-distilled H2O and drying, spots were counted under microscope.
FIGURE 1. Viral gene expression in the liver and serum ALT values in
virus-infected mice. B6 mice were injected i.v. with 2 ⫻ 109 PFU virus on
day 0, and sera was collected from groups of four mice on the days indicated for assay of ALT levels. Mice were killed and liver sections assayed
for ␤-galactosidase activity by staining. The same liver samples were assayed for ␤-galactosidase activity by an enzyme assay with similar results
(data not shown). The data shown are from one of two similar experiments.
SD values for the ALT assays are shown.
The Journal of Immunology
3037
FIGURE 4. Induction of FasL, perforin, and TNF-␣ mRNA in virusinfected mice. B6 mice were injected with 2 ⫻ 109 PFU virus on day 0,
livers were harvested on days 2 and 6, and RNA was extracted and assayed
by RT-PCR for FasL, perforin, and TNF-␣ transcripts.
FACS. Results in Fig. 3A show that in the liver but not the spleen
of infected mice there is an increase of annexin V-staining lymphocytes on days 6 and 9 after the infection. The cells that undergo
apoptosis stain with anti-CD3 Ab and are therefore T cells (Fig.
3B). It is important to note that in case of liver mononuclear cells
FIGURE 3. Appearance of apoptotic T cells in the liver of virus-infected mice. B6 mice were injected with 2 ⫻ 109 PFU virus on day 0 and
mononuclear cells isolated on the days indicated. Cells were stained for
CD3 and annexin V, then analyzed by FACS. The percentage of annexin
V-staining cells recovered from spleen and liver mononuclear cells is
shown in A. Liver mononuclear cells were stained for CD3 and annexin V.
The percentage of annexin V-staining total cells, T cells, and non-T cells
is shown in B. Data are from pooled organs from groups of three mice and
results from one of three experiments are shown.
Fas plays a role in apoptosis of CD8⫹ cells
The demonstration of apoptotic T cells in the virus-infected liver
raises questions as to the mechanism responsible for induction of
cell death. To examine which of the known apoptosis-inducing
pathways are involved, the induction of mRNA specific for
TNF-␣, perforin, and FasL was assayed in liver tissue by RT-PCR.
Results in Fig. 4 show that virus infection induces a significant
increase in message for perforin, FasL, and TNF-␣ mRNA. Therefore, Fas-, perforin-, and TNF-␣-mediated mechanisms could play
a role. To examine the possible involvement of Fas, experiments
were performed in mice lacking Fas or FasL (15, 16). B6-lpr and
B6-gld mice were infected and liver mononuclear cells analyzed
for apoptotic T cells by FACS staining. Results in Fig. 5 show that
on day 9 annexin V-staining cells are demonstrable in both the
CD4⫹ and CD8⫹ T cell populations in B6 mice. In B6-lpr and
B6-gld mice the percentage of annexin V-staining CD4⫹ cells is
identical with that in B6 mice. In contrast, annexin V-staining
CD8⫹ cells are virtually absent in both B6-lpr and B6-gld mice. It
is important to note that the majority of T cells in the virus-infected
liver are CD8⫹ cells (10, 11). Therefore, only few if any annexin
V-staining cells are demonstrable in the CD3⫹ cell population.
These results show that apoptosis of CD8⫹ cells after the viral
infection involves Fas.
FIGURE 5. Apoptosis of CD8⫹ cells is induced in normal but not in
Fas- and FasL-defective mice. B6, B6-lpr, and B6-gld mice were injected
with 2 ⫻ 109 PFU virus on day 0, and liver mononuclear cells were isolated
on day 9. Cells were stained for CD3, CD4, CD8, and annexin V, and the
percentage of annexin V-staining cells in the CD3⫹, CD4⫹, and CD8⫹ cell
population was determined. Shown are results from one of two experiments
with pooled livers from groups of three mice each.
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FIGURE 2. Allogeneic and virus-specific CTL responses in spleens of
virus-infected mice. B6 mice were injected i.v. with 2 ⫻ 109 PFU virus on
day 0, and spleens were harvested on the days indicated. A, Spleen cells
from normal (day 0) or virus-primed mice (days 10 and 28) were cultured
with irradiated BALB/c (H-2d) stimulator cells for 5 days, then assayed for
cytotoxicity on P815 (H-2d) targets at the E:T ratios indicated. SD values
for the cytotoxicity assays are shown. B, The same spleen cells were cultured with virus for 5 days and assayed for cytolytic activity on virusinfected C57SV (H-2b) targets. Shown is one of three experiments.
there is a more than 5-fold increase in the total number of cells
recovered from the liver after the infection, whereas in the spleen
it may be up to 2-fold (10, 11). Therefore, there is a significant
increase of apoptotic T cells in the liver but not the spleen of
virus-infected mice.
3038
Fas-MEDIATED ELIMINATION OF VIRUS-SPECIFIC CTL IN LIVER
FasL is expressed in liver lymphocytes pointing to cell death by
fratricide
The finding that Fas causes apoptosis of CD8⫹ cells raises the
question as to which cells in the liver express FasL and thereby
induce cell death. To examine this, use was made of bone marrow
chimeras in which the lymphoid or liver parenchyma cells express
FasL. B6-gld and B6 mice were irradiated and reconstituted with
B6 and B6-gld spleen and bone marrow cells, respectively. Infection with virus and assay for apoptotic cells reveals that the percentage of apoptotic CD4⫹ and CD8⫹ cells in B6-gld mice reconstituted with B6 cells is comparable to that of B6 mice
reconstituted with B6 cells. In contrast, in B6 mice, reconstituted
with B6-gld cells, death of CD8⫹ cells is completely inhibited
(Fig. 6A). Therefore, the apoptosis inducing FasL is not expressed
on liver parenchyma cells but rather on lymphoid cells. Among the
mononuclear cells residing in the liver are cells expressing NK cell
surface markers as well as conventional T and B cells (1, 11). To
examine which cells express FasL, mononuclear cells from livers
of virus-infected mice were analyzed by FACS. Fig. 6, B and C
shows that NK1.1⫹, CD3⫹, CD4⫹, and CD8⫹ cells all increase
expression of FasL following the infection. Therefore, death of
CD8⫹ cells could be caused by any of these cell populations. However, considering the preponderance of CD8⫹ cells in the virusinfected liver, death of CD8⫹ cells by companion FasL-expressing
CD8⫹ cells appears most likely.
Death of CD4 cells proceeds independent of Fas, perforin, and
TNF-␣ pathways and is inhibited by IL-2
Results in Fig. 5 had shown that apoptosis of CD8⫹ but not CD4⫹
cells is decreased in Fas- or FasL-deficient mice. This raises the ques-
tion why apoptosis of CD4⫹ cells is independent of the Fas mechanism and whether it can be explained by lack of Fas expression on
CD4⫹ cells. To find out, liver-infiltrating lymphocytes were stained
for CD4, CD8, and Fas expression and analyzed by FACS. Fig. 7
shows that Fas expression is identical in CD4⫹ and CD8⫹ cells isolated from normal or virus-infected livers. Therefore, the observation
that CD4⫹ cells undergo apoptosis independent of a functional Fas
pathway cannot be explained by a lack of Fas expression.
Apoptosis of CD4⫹ cells could involve the perforin or TNF-␣
rather than the Fas pathway. To examine this, perforin deficient B6PKO mice were infected with virus and liver mononuclear cells analyzed for annexin V-staining cell populations. Results in Fig. 8A
show that the percentage of apoptotic CD4⫹ and CD8⫹ cells in PKO
FIGURE 7. Expression of Fas on CD4⫹ and CD8⫹ cells. B6 mice were
infected with 2 ⫻ 109 PFU virus on day 0, and liver mononuclear cells
were harvested on day 9. Cells were double-stained for Fas, CD4, and CD8
and analyzed by FACS. The left panel shows Fas expression on CD4⫹ cells
before virus infection (solid black line to the right) and after virus infection
(dotted line to the right). The right panel shows Fas expression on CD8⫹
cells before virus infection (solid black line to the right) and after virus
infection (dotted line to the right). Results are from one of two
experiments.
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FIGURE 6. Apoptosis of T cells in bone marrow chimeras and expression of FasL on mononuclear cells from the liver. A, Bone marrow chimeras were
generated by transplanting bone marrow and spleen from B6 or B6-gld mice into irradiated B6 or B6-gld mice, as indicated. Groups of three animals each
were infected with 2 ⫻ 109 PFU virus on day 0, and livers were harvested on day 9. Mononuclear cells were isolated and stained for CD3, CD4, CD8,
and annexin V. The percentage of annexin V-staining cells in the CD3⫹, CD4⫹, and CD8⫹ cell population is plotted. B and C, Expression of FasL on
mononuclear cells from virus-infected and uninfected livers. Groups of three B6 mice were infected with 2 ⫻ 109 PFU virus each on day 0, and liver
mononuclear cells were isolated on day 9. Cells were double-stained for NK1.1, CD3, CD4, CD8, and FasL, then analyzed by FACS. Results from one
of two experiments are shown.
The Journal of Immunology
3039
FIGURE 8. T cell apoptosis proceeds in virus-infected perforin or TNF
receptor-deficient mice but not in IL-2-injected mice. A and B, B6, B6PKO, and B6-TNFR-KO mice were injected with 2 ⫻ 109 PFU virus on
day 0, and liver mononuclear cells were harvested on day 9. Cells were
double-stained for CD3, CD4, CD8, and annexin V. The percentage of
annexin V-staining cells in the CD3⫹, CD4⫹, and CD8⫹ cell population is
plotted. C, B6 mice were injected with 2 ⫻ 109 PFU virus on day 0 and
with 4 ⫻ 105 U IL-2 daily from days 0 to 8. Liver mononuclear cells were
harvested on day 9, and cells were double-stained for CD3, CD4, CD8, and
annexin V. The percentage of annexin V-staining cells in the CD3⫹,
CD4⫹, and CD8⫹ cell population is plotted. Shown are results from one of
two experiments.
mice is indistinguishable from that of normal mice. To examine the
involvement of TNF-␣, mice defective for TNFR1 and TNFR2 were
tested. Fig. 8B shows that the percentage of apoptotic CD4⫹ and
CD8⫹ cells in TNFR1/2-deficient mice is indistinguishable from that
of normal mice. Therefore, neither perforin nor TNF-␣ appear to play
a role in death of CD4⫹ cells. Apoptosis of both CD4⫹ and CD8⫹
cells likely involves an activation-induced mechanism that should be
inhibited by injection of IL-2. Indeed, results in Fig. 8C show that in
the livers of IL-2-injected mice there is a dramatic decrease of apoptotic CD4⫹ and CD8⫹ cells.
Apoptosis of CD8⫹ cells by the Fas-dependent mechanism
causes elimination of virus-specific CTL in an Ag dosedependent reaction.
The demonstration of Fas-dependent apoptosis of CD8⫹ cells in
livers of virus-infected mice suggests that the induction of unresponsiveness 4 wk after infection is the result of elimination of
virus-specific CTL (Fig. 2). For this case, one would predict reduced numbers of virus-specific CD8⫹ cells early after the infection. To examine this, the number of virus-specific, MHC class
I-restricted T cells able to secrete IFN-␥ on incubation with virusinfected targets, expressing MHC class I Ags, was assayed by
ELISPOT. Results in Fig. 9A show that spleen cells from B6 mice,
infected with virus 7 days earlier, contain only about one-third of
the IFN-␥-secreting cells, compared with spleens from B6-lpr
mice. Treatment of cells with anti-CD8 and complement before
ELISPOT assay reveals that the responding cells are predominantly CD8⫹ cells. Therefore, the Fas-mediated apoptosis mechanism reduces the number of virus-specific CD8⫹ cells in the
spleen at early times after the infection. Continued elimination of
these cells by this mechanism then accounts for the absence of
virus-specific CTL several weeks later (Fig. 2).
An interesting question is whether the induction of unresponsiveness is Ag dose dependent. To examine this, decreasing
amounts of virus were injected, followed by assay of spleen cells
for a virus-specific CTL response in vitro. Fig. 9B shows that suppression of the CTL response is virus dose dependent as higher
doses are more efficient than lower doses. These results suggest
that a high virus dose induces activation of CTL, up-regulates
FasL, and stimulates cell migration into the liver. The increase of
activated, FasL-expressing CTL in the liver should cause an increase in liver injury concomitant to an increase in Fas-dependent
apoptosis in CD8⫹ cells. Indeed, it is seen in Table I that high virus
doses stimulate expression of FasL on CD8⫹ cells, enhance migration of CD8⫹ cells into the virus-infected liver, and induce high
ALT values in the serum. In contrast, low doses, shown to prime
CTL (Fig. 9B), do not induce a significant increase in ALT values
and a much lower recruitment of CD8⫹ cells into the liver. Importantly, these CD8⫹ cells express much less FasL.
Discussion
Intravenous injection of a replication-defective adenovirus coding
for the lacZ gene causes efficient infection and viral gene expression in the liver. Numerous reports have documented that the T
cell-mediated immune system responds to virus-coded gene products, ultimately causing elimination of infected hepatocytes (8, 9,
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FIGURE 9. Decrease of virus-specific CTL at early and later times after
virus infection. A, B6 and B6-lpr mice were injected with 2 ⫻ 109 PFU
virus on day 0, and spleen cells were harvested on day 7, and restimulated
in tissue culture with virus for 5 days. Cell cultures were harvested and
assayed for IFN-␥-secreting cells on incubation with virus-infected C57SV
cells by ELISPOT. Where indicated, effector cells were treated with antiCD8 and complement before ELISPOT assay. B, Mice were injected with
different doses of virus as indicated on day 0. Spleens were harvested and
restimulated with virus on day 30. Cultures were tested for virus-specific
CTL responses at the E:T ratios indicated after 5 days. Results are from one
of two experiments.
3040
Fas-MEDIATED ELIMINATION OF VIRUS-SPECIFIC CTL IN LIVER
Table I. Fas L expression, CD8 cell migration, and serum ALT levels
are virus dose dependent
No adenovirus
5 ⫻ 108 PFU
2 ⫻ 109 PFU
5 ⫻ 109 PFU
ALT (U/L)a
CD8⫹ Cells per
Liver (⫻105)b
% FasL⫹ CD8⫹
Cellsc
25 ⫾ 20
42 ⫾ 33
645 ⫾ 42
941 ⫾ 110
5.7
13.9
88.1
109.2
0.8
3.7
9.8
13.1
a
Serum was collected on day 6 after virus infection, and ALT (units/liter) was
measured. Data are presented as mean ⫾ SD from groups of four mice each.
b
Liver mononuclear cells were isolated on day 6 after virus infection, stained with
FITC-anti-CD8 mAb, and analyzed by flow cytometric analysis. Absolute numbers of
CD8⫹ cells per liver are calculated.
c
Liver mononuclear cells were isolated on day 6 after virus infection and analyzed
by flow cytometric analysis after double-staining for FasL and CD8 as described in
Materials and Methods. Data represent percentage of both FasL⫹ and CD8⫹ cells
from liver mononuclear cells.
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17, 18). The current set of experiments was prompted by the observation that hepatocyte injury, reflected in elevated serum ALT
values decreases before a detectable decrease in viral gene expression, pointing to an active suppression mechanism that terminates
liver injury. Indeed, we were able to show that restimulated spleen
cells from mice that had received virus 28 days earlier fail to respond to a virus challenge. However, cells from these mice do
respond to allogeneic stimulator cells, leading to the conclusion
that the induction of unresponsiveness is virus specific.
In experiments aimed at elucidating the cause of unresponsiveness, it is shown that apoptotic T cells increase in the liver as early
as 6 days after the infection. But whereas both CD4⫹ and CD8⫹
cells are affected, only apoptosis of CD8⫹ cells could be demonstrated to involve the Fas pathway. This is unexpected, as a role for
Fas in apoptosis of various T cell types is well documented (19 –
21). Fas was found to control the expansion of activated T cells
during infection with Listeria, which multiplies in various organs,
including the liver (22). Fas was also reported to be responsible for
superantigen-induced deletion of CD4⫹ cells (23). Therefore, it is
unexpected that elimination of CD8⫹ but not CD4⫹ cells is attributable to the Fas mechanism. The possibility that this is caused by
lack of Fas expression was excluded by the demonstration of similar levels of Fas on both T cell types. However, consistent with the
notion that death of both CD4⫹ and CD8⫹ cells involves an activation-induced mechanism is that injection of IL-2 inhibits cell
death of both types of cells.
These results raise a number of intriguing questions. One pertains to the cells that provide activation and Fas-induced apoptosis
signals. It is well documented that despite the fact that the liver has
a relatively low expression of MHC Ags (24, 25), both MHC class
I and II molecules can be demonstrated on hepatocytes (26).
Therefore, viral epitopes may be presented on hepatocytes for recognition by CD8⫹ or CD4⫹ cells, especially after up-regulation of
MHC molecules by IFN-␥ produced by NK and T cells (11, 24).
Hepatocytes lack costimulatory receptor ligands and therefore
would be able to transmit signal 1 only, which by itself would be
paralytic and therefore lead to T cell anergy or T cell death.
That hepatocytes are able to function as APCs is demonstrated
by the fact that allogeneic hepatocytes sensitize MHC class I-specific CTL in vitro (27). However, CTL activity was found to be
limited by CTL death unless a costimulatory signal was provided
by IL-2 (27). Therefore, priming and subsequent death of CTL in
cultures stimulated by allogeneic hepatocytes may well reflect reactions responsible for T cell death in the virus-infected liver. It is
important to note here that both naive and activated T cells continuously migrate through the parenchyma and periportal field of
the liver and that activated T cells proliferate in the periportal areas
(28). Therefore, T cell death may well be induced during Ag recognition on hepatocytes in the liver.
The liver had been shown to attract activated CD8⫹ cells (29)
and proposed to serve as a graveyard for T cells undergoing Aginduced cell death (1, 30). Therefore, it is quite possible that virusspecific T cells are primed in the periphery, then migrate into the
liver where they undergo restimulation by virus-infected hepatocytes that induces apoptosis. Alternatively, T cell activation and T
cell death could be induced outside the liver, leading to accumulation of dying cells in the liver.
These considerations raise the question as to where the deathinducing FasL may be expressed. It had been shown that CD40
signaling can induce expression of FasL on hepatocytes during
chronic allogeneic liver rejection (31) and that FasL can be expressed on hepatomas (32). However, our experiments aimed at
demonstrating FasL expression on liver parenchyma cells during
induction of apoptosis in CD8⫹ cells in bone marrow chimeras
were negative. Rather, we report that viral infection up-regulates
expression of FasL on NK1.1⫹, CD4⫹ and CD8⫹ cells, pointing to
death of CD8⫹ cells by either of these cell populations. Indeed,
effector cells with respective phenotypes are well documented to
cause FasL-mediated target cell lysis (33–36). However, it seems
likely that apoptosis of CD8⫹ cells is primarily caused by CD8⫹
cells. Consistent with this, high doses of virus that are effective in
suppressing virus-specific CTL responses, induce a large increase
in CD8⫹ cells in the liver and strongly up-regulate FasL expression on these cells. Published data also support the notion that CTL
may die by Fas-mediated fratricide. Thus, incubation of CTL with
target cells able to induce expression of FasL had been shown to
induce Fas-mediated CTL death in vitro (37). Moreover, HSV 1
infection of mice causes activation-induced fratricide in virus-specific CTL via the Fas mechanisms (38). Therefore, the finding here
that CTL die by Fas-mediated and activation-dependent cell death
in the liver is not unprecedented. In contrast, it is not known why
CTL elimination in mice infected with lymphocytic choriomeningitis virus does not proceed via the Fas pathway. It is also noteworthy that despite our demonstration that TNF-␣ mRNA is induced in the virus-infected liver, no evidence for a role of this
cytokine in T cell apoptosis was obtained. Death of CD8⫹ cells by
TNF-␣ had been reported (39).
Our experiments show that the elimination of virus-specific
CTL by the Fas mechanism is Ag dose dependent, as high doses of
virus favor disappearance of primed CTL, whereas low doses promote their survival. These findings have important implications for
immune responses to allogeneic liver transplants and viral liver
infections. Liver transplants establish large numbers of MHC-expressing cells capable of inducing T cell priming as well as T cell
death. The finding in mice that after transplantation, T cells migrate into the liver and then die is consistent with a liver-induced
apoptosis mechanism (4). Also consistent is that injection of IL-2
has been reported to cause the rejection of allogeneic liver transplants (4).
Important implications from these results apply to viral infections of the liver in humans. Both HCV and HBV infections often
become persistent, leading to chronic hepatitis (6, 7). Although the
failure of the immune system to successfully eliminate virus likely
has a number of reasons, one of them may be the elimination of
virus-specific T cells. Indeed, it has been reported that in HCVinfected patients 0.1% of all T lymphocytes are eliminated in the
liver each day (40). If the majority of these T cells were to be HCV
specific, this would result in removal of effector cells destined to
eliminate the infection. Our finding here that the viral dose is a
determining factor in the elimination of virus-primed T cells is
The Journal of Immunology
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consistent with the finding that HBV-infected patients with a high
viral load have a lower frequency of virus-specific T cells, compared with patients who have eliminated the virus (41). Moreover,
drug-induced reduction of viral load in HBV-infected patients has
been shown to correlate with recovery of HBV-specific T cell responses (42). Therefore, it is quite possible that the lack of a sufficient response to infection with HBV and HCV is in part attributable to a virus dose-dependent elimination of virus-specific T
cells in the infected liver.
In summary, it is shown here that expression of foreign Ags in the
liver induces a transient cell-mediated immune response that appears
to be limited by the induction of cell death in CD4⫹ and CD8⫹ cells.
Although apoptosis of CD8⫹ cells was shown to proceed by a Fasmediated mechanism, that of CD4⫹ cells could not be demonstrated
to involve the Fas pathway. FasL is shown to be expressed on various
lymphocytes, among those CD8⫹ cells, which cause a virus dosedependent elimination of virus-specific CTL. These findings identify
an important mechanism of T cell elimination, which could in part be
responsible for acceptance of liver allografts in rodents and failure of
the cell-mediated immune system to successfully eliminate certain
viral infections in the liver.
3041