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0022-1 767/82/1296-2773$02
OO/O
THE
JOURNAL
OF
IMMUNOLOGY
Copyrlght
0
1982
by The American Assocuation
of
lmrnunologlsts
Vol
129.
No.
6,
December
1982
Prrnted
rn
U
S
A
SPECIFICITY
OF
T
LYMPHOCYTE CYTOTOXICITY TO AUTOLOGOUS HEPATOCYTES IN
CHRONIC HEPATITIS B VIRUS INFECTION: EVIDENCE THAT T CELLS ARE DIRECTED
AGAINST HBV CORE ANTIGEN EXPRESSED
ON
HEPATOCYTES’
MARIO MONDELLI, GlORGlNA MlELl VERGANI, ALFRED0 ALBERTI, DIEGO VERGANI,
BERNARD PORTMANN, ADRIAN L.
W.
F.
EDDLESTON,
AND
ROGER WILLIAMS
From the Liver Unit, the Department
of
Child Health, and the Department
of
Immunology, King’s College Hospital and Medical School,
London, England, and Centro
di
Splenoepatologia,
Istituto
de Medicina Clinica, Cattedra
di
Patologia Medica, Universita
di
Padova,
Padova, Italy
Peripheral blood T lymphocytes from 21 patients with
chronic HBV infection were incubated with autologous
hepatocytes in a microcytotoxicity assay. Cytotoxicity
was significantly increased in 13 cases, and in 12 of
these the cytotoxic effect of the
T
lymphocytes was
inhibited by preincubating the liver cells with IgG contain-
ing antibodies to the hepatitis B core antigen (HBcAg).
Normal human IgG and IgG containing antibodies to the
hepatitis
B
surface antigen (HBsAg) were without effect.
Control experiments using autologous fibroblasts as tar-
get cells showed low levels of T cell cytotoxicity and no
blocking effect of anti-core antibody.
All
patients in whom
it was possible to demonstrate HBcAg in liver tissue had
significantly increased T cell cytotoxicity to autologous
hepatocytes. These studies suggest that T cell cytotox-
icity in patients with chronic HBV infection is directed
against determinants resembling the hepatitis
B
core
antigen on the plasma membrane of hepatocytes.
It is now generally accepted that hepatitis B virus (HBV)’ is
not directly cytopathic, and that liver cell necrosis is dependent
upon the host’s immune response, possibly directed at viral
determinants on the hepatocyte membrane
(1).
There is con-
siderable evidence, including the characteristic mononuclear
inflammatory cell infiltrate present in HBsAg-positive chronic
liver disease (CLD), pointing to the importance of cell-mediated
immune mechanisms in the pathogenesis and the perpetuation
of hepatocytedamage
(2).
This hypothesis is supported by the
finding that peripheral blood lymphocytes (PBL) from patients
with chronic HBV infection are cytotoxic to a variety of target
cells
in vitro
(3-6).
Until recently, however, little effort has been
made to clarify the lymphocyte-target interaction and to char-
acterize the effector cells responsible for the
in
vitro
cytotox-
icity.
Using HBsAg-coated avian erythrocytes, Alberti and his col-
leagues
(7)
found cytolytic T lymphocytes specific for HBsAg
in patients with acute and chronic HBV infection; however, in
spite of the apparent specificity of this system, it is likely that
Received for publication May
25,
1982.
Accepted for publication August
5.
1982.
The costs of publicatlon 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.
’
Thts
work was supported by a project grant from the Medical Research
Council. Mario Mondelli was a recipient of an Anna Villa Rusconi research
fellowship.
Abbreviations used in thls paper:
HBV,
hepatitis
B
virus;
CLD,
chronic liver
disease; HBsAg, hepatitis
B
surface antigen; HBcAg. hepatitis
B
core antigen;
HBeAg. hepatitis
B
e antigen; anti-DP, antibody to the Dane particle;
NK.
natural
ktller;
LSP.
llver specific lipoprotein;
HEPES, N-2-hydroxyethylpiperazine-N’-2-
ethanesulphonic acid.
the expression of an antigen artificially coupled to xenogeneic
cells differs from its natural expression on infected human
hepatocytes.
More recently, cell lines derived from primary hepatocellular
carcinoma have been used as target cells to explore cytolytic
effector mechanisms in HBV infection. The viability of these
cells is good, allowing reproducible results to be obtained in a
conventional 5’Cr-release assay, but such studies either failed
to detect specificity of lymphocyte killing
(8)
or showed no
significant differences in cytotoxicity values between patients
and controls
(9).
These cells differ from most infected hepato-
cytes in that they do not support viral replication, and the HBV
DNA they contain is all integrated into host chromosomal
material
(10).
Further problems include the susceptibility of
most established tumor cell lines to the cytotoxic effect
of
natural killer (NK) lymphocytes
(1
1)
and restrictions on MHC
compatibility between effector and target cells, an important
requirement for optimal T cell cytotoxicity
(1
2,
13).
An alternative approach is to use autologous hepatocytes as
target cells to ensure full histocompatibility, and in our initial
studies evidence was obtained that two different effector cells
are responsible for the cytotoxic effect of PBL
in
vitro
in chronic
HBV infection, since cytotoxicity was shown to be present in
both T and non-T lymphocyte fractions
(14).
Non-T cell cyto-
toxicity seemed to be directed against autoantigens in a liver
membrane lipoprotein complex (LSP) through an antibody-de-
pendent cell-mediated mechanism
(1
4),
but the target antigen
for T cell cytotoxicity was not defined. In preliminary experi-
ments we explored the possibility that the hepatitis B surface
antigen (HBsAg) could be the target for T cell attack, but there
was a highly variable decrease in T cell cytotoxicity when
purified HBsAg was added to the cytotoxicity assay
(1
4).
This finding and the possibility that other HBV antigens
expressed on the plasma membrane of infected hepatocytes
could be target antigens for T cell cytotoxicity prompted the
series of experiments described here. Patients’ liver cells were
preincubated with monospecific and polyspecific antibodies to
HBV antigens before autologous peripheral blood T lympho-
cytes were added. The relevant antibody should cover the
corresponding viral antigen expressed on the surface of in-
fected hepatocytes and prevent target cell lysis by cytotoxic T
lymphocytes as demonstrated in other viral infections
(1
5-1
7).
MATERIALS AND METHODS
Patients.
Twenty-one HEsAg-positive patients (1
9
males and 2 females;
median age
30
yr; range
3
to
54
yr), in whom a liver biopsy was being
performed for diagnostic
or
follow-up purposes, were studied. All the
Thirteen
(62%)
of the patients had abnormal serum
aminotransferase levels
patients had been known to be HBsAg-positive in serum for at least
6
mo.
2774
HBcAg AND T CELL CYTOTOXICITY
IN
CHRONIC HBV INFECTION
(range 29 to 324 lU/liter). None of the patients had received immunosup-
pressive or antiviral drug treatment. Four patients were of British origin, the
remainder being variably distributed among populations at higher risk of
acquiring HBV infection (6 from the Middle East, 4 from the Far East, 3
African blacks, 4 from Southern Europe). Other risk factors included male
homosexuality (3) and drug addiction
(1).
cirrhosis in 10. In the remaining 9 cases the liver biopsy showed minor
Twelve patients had histologic evidence of chronic active hepatitis, with
histologic abnormalities
in
6, and chronic persistent hepatitis
in
3.
Serum samples taken at the time of biopsy were tested for HBsAg by
radioimmunoassay (RIA; Travenol Laboratories, Thetford. UK). Serum
HBeAg and anti-HBe were also tested by RIA (Abbott-HEe, Abbott Diag-
nostics, Basingstoke, UK).
Preparation
of
peripheral blood
T
lymphocytes. Fifty milliliters of blood
were taken at the time of biopsy, and leukocyte-rich plasma was obtained
by sedimentation with
5
ml of 6% (w/v) dextran in
150
mM sodium chloride
containing 600 U of preservative-free heparin. The leukocyte-rich plasma
was then layered over a Ficoll-Triosil density gradient and centrifuged at
580
X
G for
20
min (18). The interface mononuclear cells were then
incubated in a plastic petri dish for 60 min at 37OC to remove adherent cells
(1 9). Lymphocytes were subsequently sedimented by centrifugation at 300
x
G for
8
min. washed three times
in
Hanks' balanced salt solution (HBSS;
Wellcome Research Laboratories, Beckenham, UK), and resuspended in
RPMl 1640 (GIBCO Europe, Uxbridge, UK) containing 2 mM L-glutamine,
10% heat-inactivated fetal calf serum (FCS; GIBCO), 1
M
HEPES3 2.3%.
penicillin 200 U/ml, streptomycin 100 pg/ml. and amphotericin
B,
2 pg/ml,
adjusted to a pH of 7.35 (complete medium). Viability of the lymphocytes
always exceeded 98%. and less than
3%
were macrophages as assessed
by peroxidase staining and morphology (20).
A T cell-enriched subpopulation was obtained by removing the cells
bearing complement receptors after rosetting with sheep erythrocytes pre-
viously coated with the immunoglobulin fraction of anti-sheep erythrocyte
serum (Wellcome) and human complement (21
1.
The non-rosetted T cell-
enriched fraction was collected at the interface after centrifugation over a
Ficoll-Triosil densitygradient. We have previously shown that the T-enriched
fraction prepared according to this technique is contaminated with between
1 and 9% of EAC rosette-forming cells (22).
scribed previously
(22):
2 to 4 mm of human liver biopsy was collected in a
Autologous microcytotoxicity assay. This assay was similar to that de-
sterile tube containing complete RPMI-10% FCS medium and immediately
gassed with 95%
0,
5%
Con. The specimen was subsequently transferred
to a small tissue culture dish with fresh medium, gently minced with a
scalpel (Dl5 blade) into very small pieces, and exhaustively washed in
complete medium. The liver was then placed in a rotator for 4 hr at 37°C in
complete medium containing
0.01
%
collagenase (Boehringer, Mannheim,
Germany; batch no. 1449532) in an atmosphere of 95%
02
and
5%
CO,.
After being washed three times with complete medium at
50
X
G, more
than 98% of the isolated liver cells showed the ultrastructural characteristics
of hepatocytes, the remainder having the appearance of Kupffer cells.
Ten microliters of the hepatocyte suspension were then seeded into each
well of a microcytotoxicity plate (Falcon 3034F. Div. Becton Dickinson,
Grenoble, France) to achieve a final concentration of approximately 100
cells per well and were incubated at 37°C in a humidified atmosphere of
95%
O2
and
5%
CO,;
18 to 24 hr later, the supernatant was aspirated from
each well and replaced in the test chambers with the T-enriched lymphocyte
suspension adjusted to produce an effector-to-target ratio of 300:l. Con-
trols were at least
10
wells with hepatocytes plus medium alone.
After incubation at 37°C for a further 18 hr, the plates were inverted for
1 hr. gently washed with HBSS, fixed with methanol for
5
min, and stained
with 1% eosin solution. The number of hepatocytes left in each well was
counted at
X60
magnification using a graticule eyepiece. The difference
that in test wells expressed as a fraction of the former gave the percent
between the mean number of cells remaining adherent in control wells and
cytotoxicity.
The normal range was determined with T-enriched lymphocytes and
hepatocytes from 13 HBsAg negative patients with minimal changes or
normal histologic appearances on diagnostic liver biopsy. Four of these
patients had recovered from a previous episode of acute hepatitis, 2 were
thought to have Gilbert's disease,
1
Dubin-Johnson syndrome, 1 portal vein
thrombosis, 1 diabetes, 1 mild steatosis,
1
had previous granulomatous
disease,
1
had a liver biopsy before starting methotrexate therapy for
psoriasis, and finally
1
patient was referred with an increased alcohol intake
but had normal liver histology. The upper limit of normal for T cell cytotoxicity
in these cases was 33%. two standard deviations
(21)
above the mean (1
2).
experiments: (A) human anti-HBc; (B) human anti-HBe, anti-HBc;
(C)
human
Blocking experiments. The following sera were used in the blocking
anti-Dane particle (anti-DP), anti-HBc, anti-HBe: (D) goat anti-HBs ad ay
(kindly donated by Dr.
R
Tedder and Professor DS Dane, Department of
Virology, Middlesex Hospital, London); (E) normal human serum containing
no HBV markers as tested by RIA. Serum A was obtained from a patient
with HBsAg positive chronic persistent hepatitis, and sera
5
and
c
from
healthy HBsAg carriers. Serum D did not react with normal human Plasma
proteins when tested by immunodiffusion. All sera were negative for rheu-
matoid factor and antinuclear antibodies.
The IgG fraction from each serum was prepared by ion-exchange chro-
matography as described elsewhere (23). Briefly, serum was dialyzed
against four changes of starting buffer and applied to a suitable column
packed with pre-swollen DEAE-cellulose (DE-52; Whatman. Maidstone. UK)
equilibrated with the same buffer. Buffers were 10 mM sodium phosphate,
pH 8.0, for human serum and
10
mM sodium phosphate, 33 mM NaCI, pH
7.7, for goat serum. First peak fractions were pooled, concentrated, exten-
sively dialyzed against phosphate-buffered saline
(10
mM sodium phos-
phate, 150 mM NaCI). pH 7.3, and stored in small aliquots at -7OOC until
a/. (24). Each IgG preparation yielded only one band when tested by
use. The protein concentration was determined by the method of Lowry et
polyacrylamide gel electrophoresis. Anti-HBc. anti-HBs, and anti-HBe titers
were determined in each IgG preparation by RIA (Corab, Ausab. HEe,
Abbott), and antibodies to Dane particle were detected by a radioimmuno-
precipitation assay as described previously (25) (Table
I).
Further blocking
experiments were performed in two patients using monoclonal anti-HBs
antibody reacting against a common epitope in the "a" region, a generous
gift from Professor AG Siccardi, Department of Genetics, University of
Pavia. Italy. The ascitic fluid contained 10 mg/ml of specific mouse IgGl.
The IgG purification step was omitted in this case.
In the blocking procedure, 2
pl
of different dilutions of IgG were added
to at least ten microwells containing hepatocytes and medium. After incu-
bation at 37°C for 2 hr, the supernatant was aspirated from each well and,
after being washed with
10
pl
of medium, was replaced with the T-enriched
lymphocyte suspension. The experiments were then carried out as de-
scribed above.
forearm in 2 normal volunteers and 2 HBsAg-positive patients with CLD who
Preparation
of
skin fibroblast cultures. Skin biopsies were taken from the
gave informed consent for the procedure to be carried out. The skin
specimens were processed as described elsewhere (26). Briefly, skin
biopsies were cut into approximately 1-mm pieces and were placed in dry
60-mm tissue culture dishes arranged to fit a under a round 25-mm coverslip
held in place by silicone grease. Explants were then covered with
5
mi of
Eagle's modified minimum essential medium (GIBCO) containing 2 mM
L-
glutamine, 15% fetal calf serum, 1 mM sodium pyruvate,
50
U/ml penicillin,
50
pg/ml streptomycin, and 2.5 pg/ml amphotericin
8,
and were incubated
at 37°C in a humidified atmosphere of 94% air and 6%
Con.
After
10
days
the medium was removed, and the cultures were fed three times a week; 4
to 6 wk from the time the biopsies were taken, fibroblasts were harvested
by incubation at 37°C for
5
min with 0.25% trypsin in sodium citrate buffer,
pH 7.4, washed, and split further.
Only freshly trypsinized cells were used in the microcytotoxicity assay.
This assay was identical to that used with the hepatocytes as target cells.
Detection
of
HBV
core antigen
in
liver tissue. Thts was performed on
formalin-fixed paraffin-embedded liver tissue. Liver biopsy specimens were
deparaffinized and rehydrated in phosphate-buffered saline as previously
described (27). Sections were subsequently incubated with fluorescein
isothiocyanate-conjugated
anti-HBc antiserum from a healthy HBsAg carrier
for 30 min at room temperature in a moist chamber. Anti-HBc and anti-HBe
titers determined in the IgG fraction were 1
:lo'
and 1
:lo,
respectively, by
RIA. The antiserum was negative for anti-HBs by RIA. Rheumatoid factor
and antinuclear antibodies were both negative. The fluorochrome-to-protein
molar ratio was 2:1, and the protein concentration in the working dilution
was 0.25 mg/ml.
Specificity controls were performed by preincubating the sections for 30
min at room temperature with unlabeled anti-HBc antiserum and normal
human serum. Specimens were considered positive for HBcAg when a
marked reduction of the fluorescence was observed after preincubation
with anti-HBc antiserum but not with normal human serum.
Statistical analysis. The nonparametric two-tailed Mann-Whitney
U
test
for unpaired data was employed for the evaluation of the statistical signifi-
cance of differences in cytotoxicity in the experiments.
RESULTS
Thirteen
(62%)
of the
21
patients
with
HBsAg-positive
CLD
had significantly increased
T
lymphocyte cytotoxicity to autolo-
gous hepatocytes. There was
no
significant relationship be-
TABLE
l
Titers of the antiviral antibodies in the
IgG
fracbon from sera
A.
6.
C.
D,
and
E
a5
determined by
RIA
IgG Anti-HBs Anti-HBc Anti-HBe Anti-DP
A
B
-
1.3
X
105
-
-
C
-
1.6
x
lo4
1.2
x
104
-
D
-
i:8
X
io4
i:3s
X
lo3
Positive
1:lO"
E
1
:50
-
-
-
- -
-
HBcAg
AND T CELL CYTOTOXICITY IN CHRONIC
HBV
INFECTION
2775
tween the histologic findings and the degree of T cell cptotox-
icity
in
vitro (Fig. 1). The levels
of
aminotransferases were
higher in patients with raised cytotoxicity values
(90.9
k
86.4)
than in those with normal levels of cytotoxicity (44.3
f
26.51,
although this did not reach statistical significance.
When the patient's hepatocytes were preincubated with 10
to 30 pg of purified anti-HBc IgG per well (from serum A) before
addition of autologous T cells, a highly significant reduction in
T cell cytotoxicity (from 58 to 20%, mean reduction 63
f
9%
SEMI was observed in those in which cytotoxicity was initially
significantly increased (p
<
0.002) (Fig.
2).
After the preincu-
bation with anti-HBc IgG,
10
of the
13
patients tested had
cytotoxicity values within the normal range,
2
showed consid-
erable reduction, and in only
1
case was there no apparent
effect. The preincubation had no significant effect on cytotox-
icity in those patients with initial values within the normal range
(Fig. 2). Similar results (mean
%
reduction 48
-e
12 SEM) in
those with initially increased cytotoxicity were obtained using
the same concentration of IgG from serum B that was known
>
t
u
0
c
c
"
w
0
0
e
0
0
e
0
0
_"""""""""""""""
-
""_
:
8
0
0.
t
NORMAL
MINOR
CPH
CAH
UH
4
CIRRHOSIS
CONTROLS CHANGES
hepatocytes in 21 patients with chronic HBV infection grouped according to the
Figure
I.
Cytotoxicity of peripheral blood T lymphocytes for autologous
histologic appearances
on
liver biopsy. The dashed line represents the upper
limit of the normal range.
701
to contain both anti-HBe and anti-HBc antibodies (p
<
0.02).
A
smaller reduction in cytotoxicity (mean 32
&
14%
SEMI was
observed after preincubation with IgG from serum C (containing
anti-Dane particle, anti-HBc, and anti-HBe antibodies), and this
did not reach statistical significance.
The blocking effect of IgG from serum A, with anti-HBc alone,
was similar at all concentrations of IgG tested, whereas that
from sera
B
and
C
was less effective at the concentration of
10
yg/well (Table
11).
The addition of polyclonal anti-HBs IgG did
not affect cytotoxicity values (mean
%
reduction
-7
k
7
SEM)
(Fig. 21, and this finding was also confirmed in 2 cases using
monoclonal antibodies to HBsAg (mean reductions
0
and
-
10%).
Control human IgG free of detectable HBV markers
had no effect on cytotoxicity (mean
Oh
reduction 5
f
7
SEM)
(Fig. 2).
Cytotoxicity against cultured autologous skin fibroblasts.
Unfractionated PBL from both the patients tested and the
controls showed cytotoxicity against autologous skin fibro-
blasts (mean 40.5
+_
7%),
but values with T-enriched lympho-
cytes were significantly lower (mean 23
f
5.4%, p
=
0.028).
Preincubation of the fibroblasts with anti-HBc lgG did not
further reduce the cytotoxicity of the
T
cell-enriched population
for these target cells (Table
111).
In contrast, T lymphocytes from
patient MS were significantly cytotoxic to untreated autologous
hepatocytes, and preincubation of the liver cells with anti-HBc
IgG completely blocked the cytotoxic effect (from 39 to
0%).
Patient CG showed normal cytotoxicity values for autologous
hepatocytes before and after addition of anti-HBc IgG
(0
and
2%, respectively).
Detection
of
HBV
core antigen
in
liver tissue. HBcAg was
detected in the nuclei of hepatocytes of
7
patients, with a focal
TABLE
II
for
autologous hepatocytes after blocking with lgG from sera
A,
8,
and
C
at two
Mean percentage reduction m cytotoxlcity
of
peripheral blood
T
lymphocytes
different concentrations per well
Serum Source for IgG
IgG/Well
hg)
A
B
C
(anti-HBc) (anti-HBe anti- (anti-DP anti-
HBc) HBc anti-H8e)
20-30
61
%
14
57
&
14
38
2
22
(n
=
7)
(n
=
5)
10 64
A
11
(n
=
4)
32
&
23
(n
=
6) 14;O
(n
=
3)
(n
=
2)
1
Figure
2.
Mean cytotoxicity of peripheral
blood
T
lymphocytes from patients with
chronic
HBV
infection before and after block-
HEV antigens
(A-D)
and IgG from normal hu-
ing with IgG containing antibodies to different
man serum
(E).
The figures refer
to
the number
of observations. The observations for each IgG
preparation have been divided into two groups
according
to
whether lymphocyte cytotoxicity
for untreated autologous hepatocytes was nor-
mal
(-)
or significantly increased
(W).
A
B
C
D
E
IGG
USED
IN
BLOCKING EXPERIMENTS
2776
HBcAg AND
T
CELL
CYTOTOXICITY IN CHRONIC
HBV
INFECTION
pattern of distribution. Preincubation of the sections with nor-
mal human serum did not alter the intensity of fluorescence,
whereas this was markedly reduced when sections were prein-
cubated with unlabeled monospecific anti-HBc serum of com-
parable titer. All
7
patients showed significant cytotoxicity for
autologous hepatocytes, whereas only
6
of the
14
patients with
no HBcAg demonstrable in the liver had increased T cell
cytotoxicity values (p
<
0.05).
There was no relationship be-
tween the presence of HBeAg in the serum and T cell cytotox-
icity values (Fig.
3).
DISCUSSION
The finding that T cell cytotoxicity against autologous hepa-
tocytes in patients with chronic HBV infection is significantly
reduced when the liver cells are preincubated with anti-HBc
antibodies suggests that HBcAg is the principal target for T cell
cytotoxicity. However, before such a conclusion can be drawn,
it is clearly important to show that the blocking effect of the
anti-HBc IgG was due to binding
of
the antibody to the corre-
sponding antigen on the surface of infected hepatocytes. The
various control experiments were designed to test the specific-
ity
of
the blocking effect.
For
example, hepatocytes have been
shown to possess receptors for the Fc portion of IgG
(281,
and
it is conceivable that any IgG could interfere with T cell killing
by steric hindrance. However, this possibility seems unlikely in
view of the absence of any blocking effect with normal human
IgG free of detectable HBV markers.
Further evidence indicating the specificity of the blocking
effect of anti-HBc came
from
the dilution experiments using
IgG containing different amounts of anti-HBc. Thus, only the
IgG preparation containing the highest titer of anti-HBc could
effectively block the cytotoxic T cell effect at the lowest protein
concentration per well. The observation that this protective
effect for hepatocytes was only observed when T cell cytotox-
icity was significantly increased argues against the possibility
that this particular IgG contained a potent nonspecific blocking
factor. Further support for this view comes from the observation
that anti-HBc lgG did not significantly reduce the cytotoxicity
of the T-enriched population for autologous skin fibroblasts.
The finding that all cases with detectable HBcAg in liver tissue
showed significantly increased T cell cytotoxicity for autolo-
gous hepatocytes
is
in keeping with the view that core antigen
expression is associated with susceptibility to
T
lymphocyte
damage. Only formalin-fixed paraffin-embedded sections were
available as substrate for the immunofluorescence studies, and
the fixation process reduces the sensitivity for detection of
HBcAg as compared to that obtained using cryostat sections.
This reduced sensitivity may explain the absence
of
demon-
TABLE
111
cytotoxicity
of
peripheral blood lymphocytes to autologous skin fibroblasts from
norma/ controls and patients with
HBsAg
positive
CLD"
Autologous Fibroblasts Autologous Hepatocytes
Unfrac-
'E:'"
Unfrac-
Lympho-
cytes
T Cells
T
Cells Blocking
:;:",',',"_
T
Cells Blocking
after
with Anti-
HBc
IgG
cyteS with Anti-
HBc IgG
~~
Normal Controls
MM
48
26
26
RT
38
29
32
-
-
-
- -
-
Patients
MS
44
18
23
64
39
0
CG
32
19
30
30
0
2
*
The results using autologous hepatocytes from the patients are also included
for comparison
70.
50.
30
_--
lo
0
0
0
0
8
8
0
0
8
0
0'
0
0
1
00
POSITIVE NEGATl
YE
HBcAG
IN
LIVER
TlSSUE
hepatocytes isolated from patients with and without HBcAg demonstrable in liver
Figure
3.
Cytotoxicity
of
peripheral blood
T
lymphocytes for autologous
biopsy.
(0)
serum HBeAg-positive;
(0)
serum anti-HBe-positive. The dashed line
represents the upper limit
of
the normal range.
strable nuclear HBcAg in
6
of the cases with significantly
increased
T
cell cytoxicity.
Both HBeAg in the serum
(29)
and nuclear HBcAg
(30)
have
been shown to be markers of HBV replication, but it has
recently become clear that up to one-third of cases with anti-
HBe in the serum have expression of HBcAg
in
tissue sections
(31).
Similar results were obtained in the present study, and it
is of interest that increased T cell cytotoxicity
is
more closely
related to core antigen expression than to HBeAg status.
The absence of any significant blocking effect on T cell
cytotoxicity after addition of either polyclonal
or
monoclonal
antibody to HBsAg was unexpected, since HBsAg has
for
a
long time been considered the most probable target for cyto-
toxic T cells in chronic HBV infection. This observation again
emphasizes the specificity of the blocking seen with anti-HBc
IgG and suggests that HBsAg, although it may be expressed
on the plasma membrane of hepatocytes early in acute hepatitis
B
(32),
is not a target antigen for T cell attack in chronic HBV
infection, at least in this
in
vitro
system.
When cytotoxicity against virus-infected cells is detected in
any given system. it is also important to exclude the possibility
that nonspecific effector cells such as
NK
cells are responsible
for the damaging effect.
NK
cells have been shown to bear low-
affinity receptors for sheep erythrocytes
(331,
and only about
one-third have complement receptors
(34).
They may therefore
be included in the T-enriched lymphocyte population when the
purification technique employed here
is
used. However, most
NK
cells seem to possess IgG Fc receptors
(3%
and we have
previously shown that neither the additional removal of EA-
rosette forming cells nor the addition of heat-aggregated IgG
to the culture wells significantly modified T cell cytotoxicity
results
(1
4).
Furthermore, the restricted antigen specificity of
the effector cells revealed by the present blocking experiments
is a feature of T cells, not of NK cells.
The results of the experiments in which patients' and Control
lymphocytes were incubated with autologous fibroblasts were
quite different from those obtained with autologous hepato-
cytes. Here the lack of antigen specificity and the finding that
the cytotoxicity was reduced by removing the non-T fraction
suggest that the effector cells were NK, not
T
cells.
If HBcAg is the main target antigen for T cell attack in chronic
HBV infection, as it seems to be
in vitro,
it would seem likely
that infected hepatocytes would become covered with anti-HBc
antibodies, which are almost always present in the serum of
HBsAg-positive patients. It is of interest that Trevisan and his
colleagues
(36)
have shown a rather close relationship between
the presence
of
HBcAg in the nuclei of hepatocytes isolated
from HBsAg-positive patients and surface IgG deposition. Re-
cent studies from this group have also demonstrated that IgG
eluted from such liver cells contains anti-HBc but not anti-HBs
antibodies and that after elution there is a considerable in-
crease in core antigen expression on the hepatocyte rnern-
brane, as detected by immunofluorescence
(37).
In vivo
it is
possible that anti-HBc competes with cytotoxic T lymphocytes
directed against virus-infected cells and could therefore be a
modulating factor in determining the extent of the liver cell
damage. This situation is analogous to that explored by Old-
stone and Tishon
(38),
in
which they studied the modulation by
anti-measles antibody of viral antigenic expression
of
measles-
infected HeLa cells, which rendered them resistant to lysis by
sensitized effector cells. In other studies from that group it was
shown that in cells chronically infected with lymphocytic cho-
riomeningitis virus, the number of antigenic sites required to
promote T cell cytotoxicity is less than can be detected by
imrnunoflourescence
(39).
Thus, it is possible that some HBV-
infected hepatocytes could apparently be covered with anti-
HBc antibody and yet remain susceptible to T cell immune
assault. The balance between modulation of HBcAg expression
by antibody and T cell cytotoxicity may not only influence the
extent of liver damage in chronic HBV infection but could be
one of the factors determining whether there is complete clear-
ance of virus-infected cells in acute hepatitis B or progression
to persistent viral infection.
Acknowledgments.
We are indebted to Dr.
R.
P.
H. Thomp-
son and to Dr.
I.
Woolf for allowing us to study patients under
their care; to Dr.
E.
Villa for the electron microscopy studies on
isolated hepatocytes; to
Dr.
K.
Lambert for her expert advice
on tissue culture techniques; to Mrs.
Y.
S.
White for determining
the antiviral antibody titers; and to Miss Sarah Underhill for
editorial assistance.
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