CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice
et al. (2014) CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of
Acute Viral Hepatitis in Mice. PLoS ONE 9(1): e86348. doi:10.1371/journal.pone.0086348
CD4+ T-Cell Help Is Required for Effective CD8+ T Cell- Mediated Resolution of Acute Viral Hepatitis in Mice
Tanja Trautmann 0
Jan-Hendrik Kozik 0
Antonella Carambia 0
Kirsten Richter 0
Timo Lischke 0
Dorothee Schwinge 0
Hans-Willi Mittru cker 0
Ansgar W. Lohse 0
Annette Oxenius 0
Christiane Wiegard 0
Johannes Herkel 0
Jean-Pierre Vartanian, Institut Pasteur, France
0 1 Department of Medicine I, University Medical Center Hamburg-Eppendorf , Hamburg, Germany , 2 Institute of Microbiology, Swiss Federal Institute of Technology Zurich, Zu rich, Switzerland, 3 Institute of Immunology, University Medical Center Hamburg-Eppendorf , Hamburg , Germany
Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA2/2 mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the antiviral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-c production and degranulation capacity were impaired in CIITA2/2 mice. The impaired CD8+ T-cell function in CIITA2/2 mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.
Funding: The study was supported by the Deutsche Forschungsgemeinschaft (SFB 841) (http://www.dfg.de/). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Viral infections of the liver are a major cause of illness and
death worldwide. In particular, virus-induced hepatitis, leading to
chronic disease in hundreds of millions of people, is one of the
most common causes of liver cirrhosis and liver cancer . After
infection with hepatitis viruses, some individuals are able to clear
the infection, whereas others remain infected and manifest chronic
liver inflammation . The ability to clear viral liver infection is
determined both by viral and host factors, but the adaptive
antiviral immune response is believed to be the most important
determinant . Indeed, patients that spontaneously clear the
infection during acute hepatitis, show a vigorous and polyclonal
Tcell response, whereas chronically infected patients seem to have
delayed, transient or pauciclonal T-cell responses .
It is widely accepted that CD8+ T cells are the major effector
cells that mediate viral clearance from the liver by removal of
infected cells; the role of CD4+ T cells in viral hepatitis is less clear
. On the one hand, relapse of HCV infection after initial
control was associated with a loss of the antiviral CD4+ T-cell
response . Moreover, re-challenge of chimpanzees that had
cleared a previous viral infection was poorly controlled in the
absence of a functional CD4+ T-cell response [10,11].
Furthermore, several studies (reviewed in ) indicate an association
between a broad initial antiviral CD4+ T-cell response and viral
clearance. However, on the other hand, depletion of CD4+ T cells
in an early phase of HBV infection did not influence duration and
outcome of acute HBV infection in a chimpanzee study .
Moreover, recent findings indicate that the early presence of a
broad anti-HCV CD4+ T-cell response does not determine
whether HCV is cleared or persists . Furthermore, at least in
certain virus infections, type I IFN is able to promote anti-viral
CD8+ T-cell responses without dependence on CD4+ T cell help
. Thus, the role of CD4+ T cells in the early phase of viral liver
infection remains to be clarified.
To address this issue in a controlled study, we used a mouse
model of viral hepatitis induced by infection with Lymphocytic
Choriomeningitis Virus (LCMV) of the strain WE. Infection with
a high inoculum (106 FFU) of LCMV-WE causes acute hepatitis
[15,16]; the virus is usually cleared by wild-type mice within about
two weeks. LCMV hepatitis is a useful model for human hepatitis
virus infections, in so far as LCMV-WE, similar to human
hepatitis viruses, causes a non-cytopathic infection, in which the
liver damage is mediated almost entirely by the antiviral immune
response . Also in LCMV infection, CD8+ T cells are essential
for the elimination of the virus [17,18]. It is believed that CD4+ T
cells are required for sustaining CD8+ T-cell responses, thus
preventing CD8+ T-cell exhaustion and chronic LCMV infection
Figure 1. Increased and persistent LCMV infection in CIITA2/2 mice. C57BL/6 and CIITA2/2 mice were infected with 106 Focus Forming
Units (FFU) LCMV-WE. (A) At the indicated time-points after infection, spleens were homogenized and the virus titer was determined by the Focus
Forming Assay. Black dots represent the mean titer of C57BL/6 mice, white squares represent the mean titer of CIITA2/2 mice. The broken line
represents the limit of detection. (B) At the indicated time-points after infection, the infection rate of the liver was determined as in (A). (C) At the
indicated time-points after infection, the infection rate of the liver was determined by quantitative RT-PCR analysis for the LCMV Z RNA. (D) Frozen
liver sections taken at the indicated time-points after infection were stained for the LCMV nucleoprotein with VL4 antibody (green); nuclei were
stained with Hoechst 33258 (blue). (E) At the indicated time-points after infection, serum ALT levels were determined. The graphed lines represent
the mean and s.e.m.
[19,20]. Indeed, administration of CD4+ T cells can resurrect an
already exhausted CD8+ T-cell response . However, CD4+ T
cells do not seem to be required for the initiation of the CD8+
Tcell response to LCMV and the control of acute LCMV infection
To study the role of CD4+ T cells in LCMV-induced hepatitis,
we compared the outcome of LCMV infection in wild-type
C57BL/6 mice that have normal CD4+ T-cell numbers with that
in CD4+ T cell-lymphopenic C57BL/6 mice. CD4+ T
celllymphopenia was either induced by anti-CD4 antibody-mediated
cell depletion or by genetic deficiency of the major
histocompatibility (MHC) class II transactivator (CIITA) in CIITA2/2 mice
. CIITA is the master regulator of MHC class II expression in
peripheral tissues . In CIITA2/2 mice, CD4+ T cells
develop, but do not expand, as they are not appropriately
stimulated in the periphery . We demonstrate that CD4+ T
cell-lymphopenic mice manifested prolonged LCMV infection of
the liver, as compared to CD4+ T cell-replete mice. Moreover,
CD4+ T cell-lymphopenic mice manifested significantly reduced
numbers of liver-infiltrating cytotoxic CD8+ T cells. In particular,
the numbers of functional LCMV-specific CD8+ effector T cells
were significantly reduced in the livers of CD4+ T
celllymphopenic mice. The reduction of antiviral CD8+ T-cell
effector functions was not associated with elevated exhaustion
markers in the liver. These findings indicate that CD4+ T-cell help
is required to establish efficient virus control by CD8+ T cells in
the liver during acute LCMV infection.
the virus titer was determined by FFA. Each dot (black: C57BL/6 mice;
white: CIITA2/2 mice) represents the average FFU of one sample
tested in duplicate; the broken line represents the limit of detection. (B)
Livers were homogenized and the infection rate was determined as in
(A). (C) The infection rate of the livers was determined by quantitative
RT-PCR analysis for the LCMV Z RNA. (D) Serum ALT levels were
determined. (E) Frozen liver sections were stained for the LCMV
nucleoprotein with VL4 antibody (green); nuclei were stained with
Hoechst 33258 (blue).
Materials and Methods
Mice and Virus
C57BL/6 mice and CIITA2/2 mice on C57BL/6
background were bred and kept at specific pathogen-free conditions in
the animal facilities of the University Medical Centre
HamburgEppendorf. Male mice were used at the age of 8 to 15 weeks;
LCMV infection was performed at the animal facilities of the
Heinrich-Pette Institute, Leibniz Institute for Experimental
Virology, Hamburg. All animal work has been conducted
according to relevant national and international guidelines; animal
experiments were approved by the review board of the State of
Hamburg, Germany (Permit number 96/06 and 102/10).
LCMVWE was originally provided by Prof. R. M. Zinkernagel
(University Hospital, Z urich, Switzerland) and propagated on
L929 mouse fibroblasts (DSMZ-No. ACC-2). Mice were infected
intravenously with 106 FFU of virus, as determined by a modified
protocol of the previously described Focus-Forming Assay .
Here, staining of Foci was conducted with the DAKO Envision
RNA Isolation and RT-PCR
Mouse livers were snap frozen and total RNA was isolated using
a NucleoSpin TriPrep Kit (Macherey-Nagel). 1 mg of RNA was
used for reverse transcription using the AMV First-Strand
Synthesis Kit (Roche). Quantitative real time-PCR for the LCMV
Z-Protein was performed with cDNA of 0.05 mg transcribed RNA
and LightCycler FastStart DNA Master SYBR Green I (Roche).
Peptidylprolyl Isomerase A (PPIA) Primer Assay (Qiagen) was used
for normalization according to the DCt method . All PCRs
were performed in duplicate with the primer sequences
LCMV_Z_fwd (59-CAGACACCACCTATCTTGG-39) and
Alternatively, TaqMan probes for chemokines CXCL9 (Mm
00434946_m1), CXCL10 (Mm 00445235_m1), or CXCL11
(Mm 00444662_m1) were used in TaqMan PCR (Life
Depletion of CD4+ T Cells
CD4+ T cell-depleting antibody (GK1.5) and rat IgG2b
isotypematched control antibody (LTF-2) were obtained from BioXCell.
At days 3, 2 and 1 before LCMV infection, 0.3 mg of antibody
were injected intraperitoneally into C57BL/6 wild-type mice;
followed by antibody injections twice weekly after virus
Antiviral immune responses were analyzed in spleen and liver of
infected mice. Following mechanical dissection of the spleen,
splenocytes were subjected to ACK lysis. After perfusion with PBS,
livers were mechanically dissected and mononuclear liver cells
were obtained through isolation on a density gradient. To that
end, liver cells were taken up into a layer of 5 ml 30% Percoll (GE
Healthcare), covered onto a layer of 3 ml 70% Percoll, and
subjected to centrifugation at 524 g for 20 min at 20uC.
Figure 3. Reduced numbers of CD8+ T cells in the livers of LCMV-infected CIITA2/2 mice. C57BL/6 and CIITA2/2 mice were infected
with 106 FFU LCMV-WE. (A) At day 12 or 15 after infection, the numbers of CD8+ T cells in spleen and liver of C57BL/6 and CIITA2/2 mice were
determined. Each dot represents the absolute number of CD8+ T cells per spleen or liver of one individual mouse. (B) Frozen liver sections taken at
day 12 or 15 after infection were stained for CD8 T cells (red); nuclei were stained with Hoechst 33258 (blue).
Figure 4. Preserved chemokine production and non-specific leukocyte recruitment to livers of infected CIITA2/2 mice. C57BL/6 and
CIITA2/2 mice were infected with 106 FFU LCMV-WE. (A) At the indicated time-points after infection, expression of the CXCR3 chemokine ligands
CXCL9, CXCL10 and CXCL11 in infected livers were determined by qPCR. Black dots represent C57BL/6 mice, white squares represent CIITA2/2 mice.
(B) At the indicated time-points after infection, the numbers of leukocytes in infected livers were determined. Each dot represents the absolute
number of liver-infiltrating mononuclear cells of one individual mouse.
Dead cell staining was performed with Pacific Orange
Succinimidyl Ester, Triethylammonium Salt (Life Technologies)
in PBS. Immunofluorescent surface staining of T cells was
performed in 2% BSA with fluorochrome-conjugated antibodies
to CD4, CD8, CD44, CD107a, PD-1 or IFN-c (all from
BioLegend), or with Dextramers specific for the immunodominant
H-2Db restricted gp33-41 (KAVYNFATC) LCMV peptide
(Immudex). Stained cells were fixed overnight in 1% PFA in
PBS. For staining of CD107a or intracellular IFN-c, cells were
stimulated for 4 hours with the immundominant gp33-41
(KAVYNFATC) LCMV peptide (3 mg/ml) in the presence of
1 ml/ml Golgi-Plug (IFN-c) or 0.65 ml/ml Golgi-Stop (CD107a)
(both from BD Bioscience) in Panserin 401 medium (PAN Biotec),
supplemented with 1% penicillin/streptomycin and 0.56104 M
bMercaptoethanol. For intracellular IFN-c staining, cells were then
perforated in buffer containing 2% BSA/0.5% Saponin and
stained with antibody against IFN-c. Flow cytometry was
performed with an LSR II cytometer and data were analyzed
with FACSDiva 6.0 Software (both BD Bioscience).
Frozen liver sections were blocked with 1% BSA, 5% normal rat
serum, 1:50 Fc-Block (eBioscience) and 1:50 mouse IgG in PBS.
Staining was performed in 1% BSA and 5% normal rat serum
with CD8-PE (Abcam) and VL-4 antibody from a hybridoma cell
line labeled with Alexa-488 labeling kit (Life Technologies). Cell
nuclei were stained with Hoechst 33258 (Life Technologies).
Microscopy was performed with a Keyence BZ-9000 microscope.
Statistical significance of differences between two data sets was
tested by the Mann-Whitney test; for comparison of multiple
groups, the Kruskal-Wallis test and Dunns post test were
performed. P values ,0.05 (*), ,0.01 (**), ,0.001 (***) were
considered significant. Bars represent the medians.
Figure 5. Reduced numbers of LCMV-specific CD8+ T cells in the livers of LCMV-infected CIITA2/2 mice. C57BL/6 and CIITA2/2 mice
were infected with 106 FFU LCMV-WE. At day 12 or 15 after infection, the numbers of LCMV-gp33 specific CD8+ T cells in spleen and liver of C57BL/6
and CIITA2/2 mice were determined by staining with LCMV-gp33 loaded H-2Db dextramer. Each dot represents the percentage of dextramer+ CD8+
T cells among all CD8+ T cells in spleen or liver of individual mice.
Defective Clearance of LCMV from Livers of CIITA2/2
We first analyzed CIITA2/2 mice for CD4+ and CD8+ T-cell
numbers in spleen and liver in comparison to wild-type C57BL/6
mice (Figure S1). As expected, the numbers of CD4+ T cells were
greatly reduced both in spleen and liver of CIITA2/2 mice;
nonetheless, both mouse strains showed comparably high numbers
of CD8+ T cells.
We then infected wild-type C57BL/6 mice and CIITA2/2
mice with LCMV-WE at a dose of 106 FFU. At various
timepoints after infection (days 4, 7, 9, 12, 15, 18, 21 and 30), we
determined the virus titers in spleen (Figure 1A) and liver
(Figure 1B) via Focus-Forming Assay, as well as by quantitative
RT-PCR of liver RNA for the LCMV Z protein (Figure 1C). The
early infection kinetics were similar in both mouse strains;
however, at all time points from day 12 onwards, CIITA2/2
mice manifested a significantly higher degree of liver infection
than wild-type mice. Whereas wild-type mice had cleared the
infection by day 15, the infection persisted in CIITA2/2 mice for
at least 30 days. We further confirmed these findings by
histological staining of liver sections for the LCMV nucleoprotein
with VL4 antibody (Figure 1D). At all time points analyzed, there
were more VL4 positive hepatocytes in CIITA2/2 mice than in
C57BL/6 mice. Consistent with the higher and protracted viral
load, CIITA2/2 mice manifested increased and prolonged
elevation of serum ALT levels, as compared to C57BL/6 mice
(Figure 1E). Thus, numerical impairment of CD4+ T cells in acute
LCMV infection seemed to prevent the clearance of LCMV-WE
infection and to induce protracted viral hepatitis.
Defective Clearance of LCMV from Livers of
To confirm that the observed impairment of virus control in
CIITA2/2 mice was indeed caused by CD4+ T
cell-lymphopenia and not by some other effect mediated by CIITA deficiency,
we infected wild-type C57BL/6 mice that had been depleted of
CD4+ T cells with anti-CD4 antibody (GK1.5). As control,
C57BL/6 mice were treated with an isotype-matched control
antibody. The efficiency of CD4+ T-cell depletion was confirmed
by flow cytometry (Figure S2 A and B). At day 18 after LCMV
infection, we determined the virus titers in spleen (Figure 2A) and
liver (Figure 2B) by Focus-Forming Assay, as well as by
quantitative RT-PCR for the LCMV Z protein (Figure 2C).
Consistent with the results obtained in CIITA2/2 mice, we
found that CD4+ T cell-depleted mice, at day 18 after infection,
still manifested significantly higher viral titers in the liver, whereas
control IgG-treated animals had cleared the virus. Moreover,
CD4+ cell depleted mice showed overt hepatitis, as indicated by
significantly elevated serum ALT levels (Figure 2D). The
protracted infection of CD4+ T cell-depleted mice was confirmed
by histological staining of liver sections for the LCMV
nucleoprotein with the VL4 antibody (Figure 2E), clearly showing more
VL4-stained hepatocytes in CD4+ T cell-depleted mice. These
findings confirmed that the presence of CD4+ T cells during acute
LCMV hepatitis is required for effective virus control.
Impaired LCMV-specific CD8+ T cell Response in Infected
Livers of CIITA2/2 Mice
Since viral clearance is predominantly mediated by CD8+ T
cells, we next analyzed whether the inability of CD4+ T
celllymphopenic mice to control LCMV infection was associated with
altered frequencies of CD8+ T cells in the liver. We determined
the overall numbers of CD8+ T cells in infected spleen and liver of
wild-type and CIITA2/2 mice in the course of LCMV infection.
At days 7 and 9 after LCMV infection, the CD8+ T cell numbers
in the liver were equally low both in C57BL/6 mice and
CIITA2/2 mice (Figure S3). At day 12, however, CD8+ T cell
numbers in the livers of C57BL/6 greatly increased, whereas the
numbers of CD8+ T cells in the livers of CIITA2/2 mice were
significantly lower (Figure 3A). At day 15, the CD8+ T-cell
numbers were similarly low in both mouse strains; however, at this
time point, C57BL/6 wild-type mice had already cleared the
infection (Figure 3A). Of note, the CD8+ T-cell paucity seemed to
be specific to the liver, since in the spleen, CD8+ T-cell numbers
increased from day 12 to day 15 in both mouse strains. We
confirmed this finding by histological staining of CD8+ T cells in
liver sections of infected mice, showing greatly decreased numbers
of CD8+ T cells in CIITA2/2 livers at day 12 and equally low
Figure 6. Reduced IFN-c production by CD8+ T cells of CIITA2/2 mice. C57BL/6 and CIITA2/2 mice were infected with 106 FFU LCMV-WE.
At day 12 or 15 after infection, the effector function of LCMV-specific CD8+ T cells in spleen and liver of C57BL/6 and CIITA2/2 mice was determined
by intracellular staining of CD8+ T cells for IFN-c after stimulation with LCMV-gp33 peptide. Each dot represents the percentage of IFN-c+ CD8+ T
cells among all CD8+ T cells of spleen or liver of individual mice.
CD8+ T-cell numbers in wild-type and CIITA2/2 livers at day
15 after infection (Figure 3B). These findings indicated that CD8+
T cells were either ineffectively recruited to infected livers of
CIITA2/2 mice or that the liver-infiltrating CD8+ T cells were
not expanded in the absence of CD4+ T cells.
As CD8+ T cell recruitment to inflamed liver mainly depends
on the CXCR3 chemokine ligands CXCL9, CXCL10 and
CXCL11 [28,29], it was possible that the paucity of CD8+ T
cells in LCMV-infected livers of CIITA2/2 mice were due to
reduced chemokine production. We therefore analyzed expression
of these chemokines in livers of infected mice at various
timepoints (Figure 4A), but did not find major differences between the
expression levels of CXCL9, CXCL10 or CXCL11 in C57BL/6
mice and CIITA2/2 mice. Thus, CD8+ T cell paucity in
infected livers of CIITA2/2 mice was unlikely to be explained by
recruitment defects. Indeed, at various time-points after
LCMVinfection, the total numbers of leukocytes that had been recruited
to the livers of CIITA2/2 mice were at least as high as those that
had been recruited to C57BL/6 livers (Figure 4B).
To analyze the LCMV-specific CD8+ T-cell response, we used
LCMV-gp33 loaded H-2Db dextramers to detect virus-specific
CD8+ T cells by flow cytometry; a representative dextramer
staining is shown in Figure S4. At days 7 and 9, the numbers of
dextramer+ LCMV-specific CD8+ T cells were similar in the
Figure 7. Reduced degranulation capacity of CD8+ T cells of CIITA2/2 mice. C57BL/6 and CIITA2/2 mice were infected with 106 FFU
LCMV-WE. At day 12 or 15 after infection, the degranulation capacity of CD8+ T cells (A) or LCMV-specific dextramer+ CD8+ T cells (B) in spleen and
liver in response to stimulation with LCMV-gp33 peptide was determined by staining for CD107a. Each dot represents the percentage of CD107a+
CD8+ T cells among all CD8+ T cells (A) or CD107a+ dextramer+ CD8+ T cells among all CD8+ T cells (B) of spleen or liver of individual mice.
livers of infected C57BL/6 mice and CIITA2/2 mice (Figure
S5). However at day 15, we found significantly decreased numbers
of dextramer+ LCMV-specific CD8+ T cells in the spleens and
livers of CIITA2/2 mice (Figure 5), indicating that CD4+ T cells
seem to be required for efficient expansion or maintenance of
virus-specific CD8+ T cells during acute infection. We then
analyzed the antiviral effector functions of liver-infiltrating CD8+
T cells in response to stimulation with the immunodominant
LCMV-gp33 peptide. To that end, we first stained liver-infiltrating
CD8+ T cells for intracellular IFN-c in response to stimulation
with the LCMV-gp33 peptide (a representative staining is shown
in Figure S6). At days 7 and 9, there was no difference in IFN-c
response between C57BL/6 mice and CIITA2/2 mice (Figure
S7). At days 12 and 15, however, we found that CIITA2/2 mice
manifested significantly reduced hepatic frequencies of IFN-c
producing CD8+ T cells in response to LCMV-gp33 peptide
stimulation (Figure 6), which is in accordance with the finding that
CIITA2/2 mice had significantly lower numbers of
LCMVspecific CD8+ T cells. We then assessed the ability of the CD8+ T
cells to degranulate in response to stimulation with the
LCMVgp33 peptide by staining for CD107a; a representative CD107a
staining is shown in Figure S8. At days 7 and 9, there was no
difference in degranulation capacity between C57BL/6 mice and
CIITA2/2 mice (Figure S9). However, from day 12 to day 15,
CD8+ T cells from C57BL/6 mice showed a significantly
increased degranulation capacity in response to the stimulation
with LCMV-gp33 after infection (Figure 7A), which seems to
correspond with the increase from day 12 to 15 of the intrahepatic
frequency of LCMV-specific T cells (see Figure 5). In contrast,
CIITA2/2 mice exhibited a reduced number of intrahepatic cells
that were able to degranulate, both on days 12 and 15. We also
assessed the degranulation capacity of LCMV-specific dextramer+
CD8+ T cells in response to the stimulation with LCMV-gp33
(Figure 7B and Figure S8), and found that the percentage of
degranulated cells among the LCMV-specific CD8 T cells in the
liver of C57BL/6 mice was similarly high on days 12 and 15. In
contrast, the percentage of CD107a+ cells among the intrahepatic
LCMV-specific dextramer+ CD8+ T cells decreased significantly
in CIITA2/2 mice from day 12 to day 15. These findings
indicate that CD4+ T cells seem to support also the per-cell
function of LCMV-specific CD8+ T cells in the liver either directly
or indirectly, and thus contribute to a faster control of LCMV
To study whether the decreased degranulation capacity of
LCMV-specific CD8+ T cells in the livers of CIITA2/2 mice
was a consequence of T-cell exhaustion, we analyzed the
expression of the major exhaustion marker PD-1 on
LCMVspecific CD8+ T cells in the liver at day 15 after infection; PD-1 is
considered to be the first exhaustion marker expressed by
increasingly dysfunctional CD8+ T cells . However, we did
not find increased PD-1 expression in the total intrahepatic CD8+
T cell population of CIITA2/2 mice (Figure 8A) or in the
Figure 8. PD-1 expression of LCMV-specific CD8+ T cells in wild-type and CIITA2/2 mice. C57BL/6 and CIITA2/2 mice were infected with
106 FFU LCMV WE. At day 15 after infection, the expression of the exhaustion marker PD-1 on LCMV-specific CD8+ T cells in spleen and liver was
determined by staining of dextramer+ CD8+ T cells for PD-1. (A) Each dot represents the percentage of PD1+ dextramer+ CD8+ T cells among all
dextramer+ CD8+ T cells of spleen or liver of individual mice. (B) Each dot represents the mean fluorescence intensity (MFI) of PD-1 staining of all
dextramer+ CD8+ T cells in spleen or liver of individual mice.
intrahepatic LCMV-specific dextramer+ CD8+ T cell fraction
(Figure 8B), both when determined as percentage of
PD-1expressing cells among the CD8+ T cells (left panels) or as mean
fluorescence intensity (MFI) per analyzed cell (right panels). Thus,
the CD8+ T-cell dysfunction in acutely LCMV infected CIITA2/
2 mice is unlikely to be explained by T-cell exhaustion.
Although CD4+ T cells are essential for the generation of
memory CD8+ T cells  and sustained control of viral
infections, their role in the initiation of the adaptive immune
response to acute viral infection is controversial . Here, we had
used two different methods to induce CD4+ T cell-lymphopenia in
mice; one by genetic deficiency of CIITA (Figure S1), the other by
treatment with depleting antibody (Figure S2). Both models of
CD4+ T cell-lymphopenia manifested protracted viral infection of
the liver (Figures 1, 2), indicating that CD4+ T cells are crucial for
the initiation of an effective anti-viral immune response during
acute viral hepatitis. The paucity of CD4+ T cells seemed to
induce insufficient expansion of the intrahepatic CD8+ T-cell pool
(Figure 3), most notably of virus-specific CD8+ T cells (Figure 5).
Our data does not indicate whether the absence of CD4+ T cells
had caused inefficient local expansion of liver-infiltrating CD8+ T
cells or inefficient recruitment of CD8+ T cells to infected livers;
however, we did not find defective production of essential
chemokines for the recruitment into infected liver (Figure 4A) or
a general defect of non-specific recruitment of leukocytes
(Figure 4B). Nonetheless, it has been reported that the recruitment
of CD8+ T cells into infected tissues requires CD4+ T cell help
; therefore, this issue requires further investigation.
Intriguingly, the reduced expansion of virus-specific CD8+ T cells in the
liver not only caused globally impaired anti-viral effector functions,
notably production of IFN-c  and degranulation (Figures 6, 7),
but also impaired anti-viral functionality of LCMV-specific CD8+
T cells on a per-cell basis (Figure 7B). However, the impaired
functionality of virus-specific CD8+ T cells in the livers of CD4+ T
cell-lymphopenic mice did not seem to be associated with
increased expression of the exhaustion marker PD-1 (Figure 8);
PD-1 is the first exhaustion marker to be expressed in a
progressing loss of function  and expression is linked to viral
load . This finding indicated that functional impairment of
CD8+ T cells may occur without manifest up-regulation of
exhaustion markers, at least during the time-points analyzed in this
Our demonstration of a causal relationship between the
presence of CD4+ T cells and the outcome of acute viral liver
infection is in full agreement with previous findings that had
uncovered an association between a broad and strong CD4+
Tcell response to hepatitis viruses and the ability to clear the
infection . However, our findings seem to contradict the
findings by Thimme et al.  who showed that depletion of
CD4+ T cells does not significantly influence the clearance of
HBV infection in chimpanzees. We think that these discrepant
results can be explained by the timing of CD4+ T-cell depletion.
Indeed, in the study by Thimme et al. the CD4+ T cells were
depleted at day 6 after HBV inoculation, when the initial priming
of CD8+ T cells probably had already occurred. In our study, by
contrast, the mice were already depleted of CD4+ T cells at the
time of virus inoculation and the priming of the CD8+ T-cell
response took place in absence of CD4+ T cells.
It is generally believed that CD4+ T cells are not required for
the control of acute low-dose LCMV infections (reviewed in ),
as type I IFN can substitute CD4+ T-cell help in the priming of
LCMV-specific CD8+ T cells [14,35]. Indeed, Matloubian et al.
 had previously shown that depletion of CD4+ T cells does not
significantly influence the clearance of acute infection with LCMV
of the strain Armstrong that does, however, not cause hepatitis.
Here, in contrast, we infected mice with LCMV-WE, which is
more virulent and thus able to induce acute hepatitis . Thus, it
is possible that the differential requirement for CD4+ T cells may
be related to differential virulence of the LCMV strains. Be that as
it may, in the hepatitis-inducing LCMV-WE infection that we
have studied here, CD4+ T cell-lymphopenia seemed to induce an
impaired intrahepatic CD8+ T-cell response and impaired
intrahepatic virus control. Indeed, this functional impairment
seemed to be more distinct in the liver than in the spleen (Figure 7),
indicating that virus control in the liver depends critically on
CD4+ T-cell help.
Taken together, our findings indicate the relevance of CD4+ T
cells for virus control in the liver, also during acute viral hepatitis.
Our results suggest that ineffective CD4+ T-cell help may promote
the evolution of acute liver infection towards chronic viral
hepatitis. Therapeutic and vaccine strategies aimed at
strengthening acute CD4+ T-cell responses may hence prove useful to
prevent the chronification of hepatitis virus infections.
Figure S1 Reduced numbers of CD4+ T cells, but not
CD8+ T cells in CIITA2/2 mice. Spleen and liver
mononuclear cells of C57BL/6 wild-type and CIITA2/2 mice
were isolated and stained for CD4+ and CD8+ T cells respectively.
Each dot represents the absolute number of CD4+ or CD8+ T
cells per spleen or liver of individual mice.
Figure S2 Efficacy of CD4+ T-cell depletion by anti-CD4
antibody treatment. C57BL/6 mice were treated twice weekly
with depleting anti-CD4 antibody (GK1.5) or isotype-matched
control antibody and depletion efficacy was determined by flow
cytometry. Shown are the percentages of CD4+ T cells (stained
with anti-CD4 antibody of clone RM4-4) among all CD3+ T cells
(A) and representative CD4+ staining of individual mice at day 18
of infection (B).
Figure S4 Analysis of LCMV-specific CD8+ T cell
response with LCMV-gp33 loaded H-2Db dextramers.
LCMV-infected C57BL/6 or CIITA2/2 mice were assessed for
LCMV-specific liver-infiltrating CD8+ T cells that recognize the
immunodominant gp33 peptide bound to H-2Db molecules by
immunofluorescent staining with gp33 loaded H-2Db dextramers,
as assessed by flow cytometry. Shown are representative dextramer
stainings of liver-infiltrating CD8+ T cells from mice at day 15 of
Figure S5 Liver-infiltrating LCMV-specific CD8+ T cell
numbers in early LCMV infection. LCMV-infected C57BL/
6 or CIITA2/2 mice were assessed for LCMV-specific
liverinfiltrating CD8+ T cell numbers by immunofluorescent staining
with gp33 loaded H-2Db dextramers. Each dot represents the
percentage of dextramer+ CD8+ T cells among CD8+ T cells per
liver of one individual mouse at day 7 or 9 after LCMV-infection.
Figure S6 Analysis of IFN-c production by CD8+ T cells
in response to stimulation with LCMV-gp33 peptide.
Liver-infiltrating CD8+ T cells of LCMV-infected C57BL/6 or
CIITA2/2 mice were assessed by flow cytometry for IFN-c
production in response to stimulation with the immunodominant
LCMV-gp33 peptide. Shown are representative intracellular
IFNc stainings of liver-infiltrating CD8+ T cells from mice at day 15 of
Figure S7 Analysis of IFN-c production by CD8+ T cells
in early LCMV-infection. Liver-infiltrating CD8+ T cells of
LCMV-infected C57BL/6 or CIITA2/2 mice were assessed by
flow cytometry for IFN-c production in response to stimulation
with the immunodominant LCMV-gp33 peptide. Each dot
represents the percentage of IFN-c stained infiltrating CD8+ T
cells per liver of one individual mouse at day 7 or 9 of infection.
Figure S8 Analysis of degranulation capacity of
LCMVgp33 specific CD8+ T cells based on CD107a staining.
Liver-infiltrating LCMV-specific CD8+ T cells of LCMV-infected
C57BL/6 or CIITA2/2 mice were assessed by flow cytometry
for LCMV-gp33 loaded H-2Db dextramers (upper panels). The
dextramer+ cells were consecutively gated for CD107a staining as
degranulation marker (lower panels). Shown are representative
dextramer and CD107a stainings of liver-infiltrating CD8+ T cells
from mice at day 15 of infection. The indicated percentage of
Figure S9 Analysis of degranulation capacity of CD8+ T
cells in early infection. At day 7 or 9 after infection, the
degranulation capacity of liver-infiltrating CD8+ T cells (A) or
liver-infiltrating LCMV-specific dextramer+ CD8+ T cells (B) in
response to stimulation with LCMV-gp33 peptide was determined
by staining for CD107a. Each dot represents the percentage of
degranulated CD8+ T cells among all CD8+ T cells (A) or among
all dextramer+ CD8+ T cells (B) per liver of one individual mouse
at day 7 or 9 of infection.
We thank our colleagues from the Heinrich-Pette Institute, Leibniz
Institute for Experimental Virology for support and housing the murine
infection experiments. We thank Birte Hanisch, Marko Hilken and Agnes
Malotta for technical assistance.
Conceived and designed the experiments: TT CW JH. Performed the
experiments: TT J-HK AC TL DS. Analyzed the data: TT J-HK AC KR
TL DS H-WM AWL AO CW JH. Contributed reagents/materials/
analysis tools: KR TL H-WM AO. Wrote the paper: TT TL H-WM AWL
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