Sustained Dysfunction of Antiviral CD8+ T Lymphocytes after Infection with Hepatitis C Virus

Abstract

Hepatitis C virus (HCV) sets up persistent infection in the majority of those exposed. It is likely that, as with other persistent
viral infections, the efficacy of T-lymphocyte responses influences long-term outcome. However, little is known about the
functional capacity of HCV-specific T-lymphocyte responses induced after acute infection. We investigated this by using major
histocompatibility complex class I-peptide tetrameric complexes (tetramers), which allow direct detection of specific CD8+T lymphocytes ex vivo, independently of function. Here we show that, early after infection, virus-specific CD8+ T lymphocytes detected with a panel of four such tetramers are abnormal in terms of their synthesis of antiviral cytokines
and lytic activity. Furthermore, this phenotype is commonly maintained long term, since large sustained populations of HCV-specific
CD8+ T lymphocytes were identified, which consistently had very poor antiviral cytokine responses as measured in vitro. Overall,
HCV-specific CD8+T lymphocytes show reduced synthesis of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) after stimulation
with either mitogens or peptides, compared to responses to Epstein-Barr virus and/or cytomegalovirus. This behavior of antiviral
CD8+ T lymphocytes induced after HCV infection may contribute to viral persistence through failure to effectively suppress viral
replication.

Full text

JOURNAL OF VIROLOGY,
0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.12.5550–5558.2001
June 2001, p. 5550–5558 Vol. 75, No. 12
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Sustained Dysfunction of Antiviral CD8
⫹
T Lymphocytes after
Infection with Hepatitis C Virus
NORBERT H. GRUENER,
1
FRANZISKA LECHNER,
2
MARIA-CHRISTINA JUNG,
3
HELMUT DIEPOLDER,
3
TILMAN GERLACH,
3
GEORG LAUER,
4
BRUCE WALKER,
4
JOHN SULLIVAN,
5
RODNEY PHILLIPS,
2
GERD R. PAPE,
1,3
AND PAUL KLENERMAN
2
*
Institute for Immunology, D-80336 Munich,
1
and Medical Department II, Klinikum Grosshadern, D-81366 Munich,
3
Germany; Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
2
;
Infectious Diseases Unit and AIDS Research Center, Massachussets General Hospital and Harvard Medical
School, Boston, Massachusetts 02129
4
; and Australian Red Cross Blood Service, Sydney 2000, Australia
5
Received 23 January 2001/Accepted 12 March 2001
Hepatitis C virus (HCV) sets up persistent infection in the majority of those exposed. It is likely that, as with
other persistent viral infections, the efficacy of T-lymphocyte responses influences long-term outcome. However,
little is known about the functional capacity of HCV-specific T-lymphocyte responses induced after acute in-
fection. We investigated this by using major histocompatibility complex class I-peptide tetrameric complexes
(tetramers), which allow direct detection of specific CD8
ⴙ
T lymphocytes ex vivo, independently of function.
Here we show that, early after infection, virus-specific CD8
ⴙ
T lymphocytes detected with a panel of four such
tetramers are abnormal in terms of their synthesis of antiviral cytokines and lytic activity. Furthermore, this
phenotype is commonly maintained long term, since large sustained populations of HCV-specific CD8
ⴙ
T lymphocytes were identified, which consistently had very poor antiviral cytokine responses as measured in
vitro. Overall, HCV-specific CD8
ⴙ
T lymphocytes show reduced synthesis of tumor necrosis factor alpha
(TNF-␣) and gamma interferon (IFN-␥) after stimulation with either mitogens or peptides, compared to
responses to Epstein-Barr virus and/or cytomegalovirus. This behavior of antiviral CD8
ⴙ
T lymphocytes
induced after HCV infection may contribute to viral persistence through failure to effectively suppress viral
replication.
Hepatitis C virus (HCV) infects approximately 170 million
people worldwide and is a significant cause of liver failure. The
virus is able to set up chronic infection in about 80% of those
infected, although a minority are able to clear the virus from
blood after acute exposure. Although dynamic studies of in-
fection are hampered by the lack of acute symptoms in most
patients, there is nevertheless accumulating evidence that mul-
tiple immune responses are involved (8, 11, 19, 21, 24).
A role for CD4
⫹
T-helper lymphocytes is supported by func-
tional and genetic linkage studies (11). (7, 25, 28, 30). Re-
cent studies of acute disease have also indicated that antiviral
antibody responses directed against envelope proteins (E2)
influence viral evolution and disease outcome (9). CD8
⫹
T
lymphocytes specific for HCV also arise early after infection
(19–21) and, by analogy with hepatitis B virus infection, theo-
retically could suppress viral replication by secretion of anti-
viral cytokines (13). However, these populations are not sus-
tained and generally become difficult to detect in blood,
especially when techniques that rely on detection of gamma
interferon (IFN-␥) are used (29), although they can be recov-
ered upon stimulation in vitro (4, 17, 27, 32). There is evidence
from murine models that failure of T-cell responses to control
early-stage infection can lead to shifts in immune-selective
forces and viral escape (6, 16). We therefore asked whether a
specific dysfunction of antiviral CD8
⫹
T lymphocytes may oc-
cur after infection with HCV and addressed this question by
analyzing T-lymphocyte responses directly ex vivo with pep-
tide-major histocompatibility complex (MHC) class I tetram-
ers (1, 3, 12) combined with assays for lymphocyte function.
MATERIALS AND METHODS
Patients and blood samples. Patients were recruited from clinics in Munich,
Germany; Oxford, United Kingdom; Sydney, Australia; and Boston, Mass. Pe-
ripheral blood mononuclear cells (PBMCs) were prepared and frozen as previ-
ously described(19–21). For assays comparing different time points from the
same individual, samples were thawed and stained simultaneously. Informed
consent for venipuncture was obtained in all cases. For subjects A and B (both
HLA-A*0201-positive female intravenous drug users who presented acutely and
are studied here in the most detail), seroconversion to anti-HCV antibody pos-
itivity was determined at the time of presentation. Liver histology was not
obtained for subjects A and B, because it was not indicated in acute disease.
Their clinical details are shown in Table 1. Of the other subjects, not studied here
during acute infection, subject C also acquired virus via intravenous drug use,
subject D probably acquired the virus via sexual contact, and subject E acquired
the virus via infected blood products (subjects 1 and 3 and 15 in reference 19).
The time of acquisition of virus was determined from clinical history and from
transfusion data. The full details of the cohorts from which these subjects were
selected, including age, tissue type, sex, PCR and treatment status, and, where ap-
propriate, genotype and liver histology, have been published previously (19–21).
Class I-peptide tetramers. Class I-peptide tetramers were prepared and vali-
dated exactly as previously described. The following peptides were obtained from
Research Genetics (Huntsville, Ala.): NS3 peptide 1073–1081 (abbreviated here
as NS3 1073; CINGVWCTV), NS5B peptide 2594–2602 (NS5 2594; ALYDV
VTKL), NS4B 1406–1415 (NS4 1406; KLVALGINAV), and NS4B 1807–1816
(NS4 1807; LLFNILGGWV). The tetramers for the control Epstein-Barr virus
(EBV) and cytomegalovirus (CMV) peptides restricted by HLA-A2 (EBV
BMLF-1 [GLCTLVAML] and CMV pp65 [NLVPMVATV]) were validated by
using peripheral blood samples from EBV- and CMV-seropositive, healthy in-
* Corresponding author. Mailing address: Nuffield Department of
Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United
Kingdom. Phone: 01865 221335. Fax: 01865 220993. E-mail: klener
@molbiol.ox.ac.uk.
5550

dividuals as described previously (19, 21). In screening the HCV patients, where
appropriate, tetramers for two HCV epitopes restricted by HLA-B8 (HSKKKL
DEL and LIRLKPTL) and two restricted by HLA-B7 (GPRLGVRAT and
DPRRRSRNL) were also used, as previously described (19, 20). However, since
these did not yield large tetramer-positive populations amenable to functional
analysis, they are not detailed further.
Tetramer staining and functional assays. The following conditions were used
for staining. From 0.5 to 1 million PBMCs were coincubated with tetramer for 20
min at 37°C, followed by washing and phenotypic staining or stimulation. The
following monoclonal antibodies (MAbs) were used: anti-CD8-PerCP, anti-
CD27–fluorescein isothiocyanate (FITC) conjugate, anti-CD45RO–allophyco-
cyanin (APC), anti-CD45RA–FITC (all Becton Dickinson Immunocytometry
Systems), anti-CC chemokine receptor 5 (CCR-5)–FITC, anti-CD38–PC, anti-
CD69–APC (all PharMingen), anti-human MHC class II–FITC (HLA-DR,
-DP, and -DQ; Dako), anti-Ki 67–FITC (Coulter Immunotech, Marseille,
France), anti-perforin–FITC (Pharmingen), and FITC-conjugated anti-T-cell re-
ceptor (TCR) (TCR V␤1, -2, -3, -5.1, -5.2, -8, -12, -13.1, -14, -16, -17, -20, and -22;
unlabeled anti-TCR V␤5.3, -9, and -23 [all Coulter Immunotech]). Unlabeled
anti-TCR V␤antibodies were detected with FITC-conjugated antimouse immu-
noglobulin (Ig) [F(ab⬘)
2
; Biosource, Camarillo, Calif.]. Phorbol myristate acetate
(PMA)-ionomycin stimulation was performed exactly as described in reference
21, but the tetramer staining was done first (20 min), and then a PMA-ionomycin
mixture was added without washing. Determination of peptide stimulation and
intracellular cytokine secretion was performed in the presence of 10 ␮M cognate
peptide or control and 1 ␮g of costimulatory anti-CD28 (Pharmingen, San
Diego, Calif.) and anti-CD49b antibodies (Becton Dickinson, San Jose, Calif.)
per ml (6 h). Peptide stimulation and CD69 staining were performed in the
absence of costimulation (4 h). Flow cytometric analysis was performed with a
Becton Dickinson FACSCalibur fluorescence-activated cell sorter (FACS), and
analysis was performed with CellQuest software.
CD4 proliferative assays. CD4 proliferative assays were performed with re-
combinant HCV proteins and tritium incorporation exactly as previously de-
scribed (11).
RESULTS
Analysis of CD8
ⴙ
T-lymphocyte responses ex vivo after
acute HCV infection. We first tracked T-lymphocyte responses
in a set of four HLA-A*0201-positive patients for whom a clear
date of onset of infection was available (Table 1). Two exam-
ples (subjects A and B) in which samples were obtained very
close to the time of acquisition of virus are illustrated in detail
in Fig. 1a and described in Table 1. We analyzed CD8
⫹
T-
lymphocyte responses against four HLA-A2-restricted HCV
peptides by using peptide-MHC class II tetramers, as previ-
ously described (Fig. 1a, lower panel) (19, 21). In subject A, a
significant response was observed against NS3 1073 (CINGVW
CTV [shown in Fig. 1a, middle panel]) and NS5 2594 (ALYD
VVTKL), but not NS4 1406 (KLVALGINAV) or NS4 1807
(LLFNILGGWV) (not shown). In subject B, only a response
to NS3 1073 was observed, but in this instance, we also had the
opportunity to compare this response with established memory
responses to HLA-A2-restricted epitopes from either EBV
(GLCTLVAML) or CMV (NLVPMVATV [0.74 and 0.25% of
CD8
⫹
lymphocytes, respectively]).
Dynamics and phenotype and of antiviral CD8
ⴙ
T-lympho-
cyte responses over time. Interestingly, in subject A, a shift in
dominance between the two detectable responses was noted
over time. This occurred during a period in which the subject’s
blood was already negative for viral RNA by PCR, and a very
similar pattern was observed under similar circumstances in a
previously described case (21), as well as in cases later after
infection (19, 21). The shift in numerical dominance was par-
alleled by a striking shift in expression of markers of activation
between the two populations (Fig. 1b). The response, which
peaked second (directed against NS5 2594), showed peak
CD38 and class II expression 2 weeks later than the first wave
of cells directed against NS3 1073. Also, both populations
showed increased expression of chemokine receptor CCR5 at
the time of their maximal activation, which might contribute to
recruitment into the inflamed liver. The level of expression of
the intracellular marker of proliferation Ki-67 was high for the
first wave of cells observed, at a time when their numbers in
blood were actually waning. This is consistent with either ex-
haustion of this response or redistribution into the liver.
Analysis of function of tetramer-positive cells ex vivo. We
next examined the function of these virus-specific CD8
⫹
T-
lymphocyte populations ex vivo by using an established assay
for intracellular detection of IFN-␥synthesis after stimulation
with PMA-ionomycin (21). These are illustrated in Fig. 1c to e
in detail for subject B, for whom an internal comparison was
available. In this individual, the level of IFN-␥synthesis after
stimulation was low in the HCV-specific tetramer-positive pop-
ulation and was significantly lower at both time points tested
than those in the control CMV- and EBV-specific populations
(Fig. 1c). The release of IFN-␥in response to peptide stimu-
lation (a more physiological stimulus, since triggering occurs
through the TCR) showed even more profound defects (Fig.
1d) compared to the internal control of the CMV-specific
response. This dysfunction was sustained over a period of 3
months. Consistent with this, a highly sensitive overnight ex
vivo IFN-␥ELISpot assay (Mabtech, Upsalla, Sweden) per-
formed with the same NS3 peptide was also completely nega-
tive throughout this period (data not shown) (18). Intermedi-
ate time points over this period showed a very similar profile of
unresponsiveness. For example, at week 7 in subject B, at the
peak of the CD4 responses (Fig. 1a, lower panels), the IFN-␥
ELISpot remained negative for responses against NS3 1073, as
was synthesis of tumor necrosis factor alpha (TNF-␣) after
PMA stimulation.
Analysis of CD69 upregulation in comparison to control
responses. To confirm that the defect in cytokine synthesis
represented a failure of triggering (rather than a switch in
secretion pattern to other cytokines), upregulation of CD69
after NS3 1073 peptide stimulation in vitro was also measured
at these time points, as previously analyzed (21). Again, weak
responses were observed, most obviously when compared with
the internal CMV control (Fig. 1e and f).
Examination of tetramer-positive responses of different
specificities and in different individuals. The specificities of
TABLE 1. Clinical details of subjects studied during or after acute infection
Subject Sex Age (yr) Time of analysis HLA class I Clinical outcome
A Female 31 During acute hepatitis A2, A26, B38, B50 Relapse
B Female 37 During acute hepatitis A1, A2, B8, B40 Sustained PCR negative in blood
C Male 42 After acute hepatitis A2, A26, B37 Sustained PCR negative in blood
D Female 32 After acute hepatitis A2, A3, B41, B44 Sustained PCR negative in blood
VOL. 75, 2001 CYTOKINE RELEASE BY HCV-SPECIFIC CD8
⫹
T LYMPHOCYTES 5551

FIG. 1. Dynamics of hepatitis and acute immune responses in two subjects. (a) Time course of disease and immune responses. (Upper panels)
Time course of alanine aminotransferase (ALT [international units per milliliter]) in serum over time. Subject A was PCR positive (⫹) for HCV
RNA at the first time point and subsequently became PCR negative (⫺) over time as indicated, with recrudescence at week 15. In subject B, virus
did not recrudesce, even during longer follow-up periods of 1 year. (Middle panels) Dynamics of tetramer-positive responses. Frozen PBMCs were
thawed and tested in parallel with tetramers for four HLA-A2-restricted peptides (NS3 1073, NS3 1406, NS4B 1807, and NS5B 2594). Thawed
PBMCs were stained exactly as previously described (19, 21). Only positive stains are shown. The proportions were calculated after gating on live
CD8
⫹
lymphocytes. (Lower panels) Fresh PBMCs were tested in standard proliferation assays by incorporation of [
3
H]thymidine after stimulation
with HCV antigens as previously described (11). (b) Phenotype of acute responses in subject A. Frozen PBMCs were thawed and stained in parallel
with the tetramers for NS3 1073 and NS5 2094, shown to be positive, together with the antibodies for MHC class II CD38, CCR-5, or (after
permeabilization) Ki-67 (see Materials and Methods). The proportions of tetramer-positive cells staining positive at each time point for each
marker are shown. (c) PMA-ionomycin-stimulated cytokine synthesis over time in subject B. Frozen PBMCs were thawed, tetramer stained for
20 min, and then stimulated with PMA-ionomycin as previously described (21). Staining with PerCP-labeled anti-CD8 was followed
by permeabilization as in panel b and intracellular staining with FITC–anti-IFN-␥(Becton Dickinson). After four-color flow cytometry, the
proportion of tetramer-positive cells staining positive for intracellular IFN-␥was calculated. (d) Peptide-stimulated synthesis of IFN-␥in subject B:
comparison of HCV and control response. Frozen PBMCs from subject B at weeks 2 and 14 were thawed, stained with tetramers for HCV NS3 1073
5552 GRUENER ET AL. J. VIROL.

or CMV, stimulated with the appropriate peptide and costimulatory molecules, permeabilized, and stained for CD8 and intracellular IFN-␥(21). After
flow cytometric analysis, the CD8
⫹
population is displayed. The proportion of tetramer-positive cells staining positive for IFN-␥is shown. Staining
of cells in the absence of peptide revealed stimulation of ⬍2% in both cases. (e) Peptide-stimulated upregulation of CD69 in subject B: comparison
of HCV and control responses. PBMCs from the same time points as in panel d above were stimulated in the same manner with peptide (21) and
stained thereafter with PerCP–anti-CD8 and FITC–anti-CD69. The proportion of tetramer-positive cells expressing CD69 is illustrated. Expression
in ex vivo samples or in the absence of peptide was ⬍2%. (f) Example of CD69 upregulation in tetramer-positive populations by peptide stimulation.
Examples from the first time point of CD69 surface staining in tetramer-positive cells. The tetramer-positive CD8
⫹
population was gated upon and
CD69 expression was analyzed after peptide stimulation. No upregulation of CD69 on tetramer-negative cells was observed (data not shown).
VOL. 75, 2001 CYTOKINE RELEASE BY HCV-SPECIFIC CD8
⫹
T LYMPHOCYTES 5553

tetramer-positive responses in different subjects were repro-
duced through analyses of other responses in other patients
and other epitopes. Figure 2a illustrates cytokine staining pat-
terns in three separate individuals in whom three distinct epi-
topes (NS3 1073, NS3 1406, and NS5 2594) from HCV were
targeted. In these cases, little or no cytokine synthesis was
measured after maximal stimulation with PMA-ionomycin.
This was clear by measurement of IFN-␥and TNF-␣. These
FIG. 2. Functional analysis of HCV-specific responses against three separate epitopes in three separate donors. (a) Cytokine release from
CD8
⫹
lymphocytes of different specificities. Frozen samples from three separate HCV antibody-positive individuals were thawed and tested for
synthesis of antiviral cytokines after PMA-ionomycin stimulation exactly as in Fig. 1c. Both stains for TNF-␣(right panels [marked FL4-H]) and
IFN-␥(left panels [ifngFITC]) are illustrated. The numbers shown in each plot represent the percentage of tetramer-positive cells that stained
positive for the particular cytokine. PCR-ve, PCR negative. (b) Control unstimulated cells and a reference for gating on the CD8 high population.
Samples obtained from subject C (NS3 1073 specific) are illustrated. No synthesis of IFN-␥is seen. (c) Comparison of PMA-ionomycin and peptide
stimulation. A CMV-specific response from a control HCV-negative patient is shown. The samples were tested in parallel for TNF-␣synthesis
according to the peptide stimulation and PMA stimulation protocols, and the CD8 high population is shown. Approximately similar proportions
of CD8
⫹
cytokine-positive cells were obtained, as indicated in the right upper quadrant of each FACS plot. (d) Analysis of V␤usage of tetramer-
positive cells in subjects A and B. PBMCs were anti-CD8 and tetramer stained as described above and costained with a panel of FITC-conjugated
V␤-specific MAbs (Immunotech, Marseille, France). Staining for V␤3 only is shown, after gating on live CD8
⫹
lymphocytes. A large population
of V␤3-positive, tetramer-positive lymphocytes is seen in subject B (30% of tetramer-positive cells), and a smaller population is seen in subject A
(5%). No dominant V␤usage was seen in subject A, C, or D. Tetramer-negative populations in these subjects did not reveal a major oligoclonal
expansion when this restricted panel of antibodies was used (V␤1, -2, -3, -5.1, -5.2, -5.3, -8, -9, -12, -13.1, -14, -16, -17, -20, -22, and -23).
5554 GRUENER ET AL. J. VIROL.

individuals were all PCR negative at the time of study and had
been so for many months. The plots illustrated demonstrate
the extremes of the range of cytokine responses seen among
HCV-specific CD8
⫹
lymphocytes. In all cases, the proportion
of cytokine-positive cells among the tetramer-positive popula-
tion was lower than that in the tetramer-negative CD8
⫹
pop-
ulation. Exactly the reverse result is seen in the case of CMV-
and EBV-specific tetramer populations (described below; Fig.
3b). Again, no or very low levels of synthesis were also ob-
served after peptide stimulation (data not shown).
A series of control experiments were performed to confirm
that these results were not artifactual due to stimulation of T
cells by tetramers or downregulation effects due to stimulation.
The tetramer-positive controls left unstimulated for the dura-
tion of the assay are illustrated in Fig. 2b, which demonstrates
that staining with the tetramer alone does not lead to synthesis
of cytokines with this protocol. A typical control stain directly
comparing PMA and peptide-stimulated responses is also
shown (Fig. 2c), which demonstrates that the populations of
CD8
⫹
cells that make cytokine after peptide stimulation are
comparable with both stimuli. This experiment also shows that
the number of tetramer-stained cells remains high in phyco-
erythrin (PE) fluorescence under this assay system. Internal-
ization of tetramer occurs normally within a few minutes of
staining (31), and strong signals appear to be intact at the end
of the assay in both cytokine-positive and cytokine-negative
FIG. 3. Functional analysis of a long-term antiviral CD8
⫹
T-lymphocyte response. (a) Peptide-stimulated synthesis of cytokines in subject E:
comparison of HCV and control responses. Experiments were performed exactly as in Fig. 1c. The CD8
⫹
population is shown, and the proportions
within the tetramer-positive populations that stain positive for intracellular cytokine are indicated. The upper panels represent stimulation with
HCV peptide NS3 1073 (also indicated as “peptide 11” in the FACS plot title line), and the middle and lower panels represent HLA-A2-restricted
peptides from CMV and EBV, respectively. (b) PMA-ionomycin-stimulated TNF-␣synthesis in patient E. Experiments were performed exactly
as in Fig. 2a. The CD8
⫹
population is shown, and the proportions within the tetramer-positive and tetramer-negative populations positive for
intracellular cytokine are indicated.
VOL. 75, 2001 CYTOKINE RELEASE BY HCV-SPECIFIC CD8
⫹
T LYMPHOCYTES 5555

subsets with this protocol. This experiment also demonstrates
the lack of cytokine secretion by tetramer alone in the presence
of costimulatory antibodies.
Analysis of TCR V␤usage in acutely expanded HCV-specific
CD8
ⴙ
T-lymphocyte populations. We addressed whether the
HCV-specific CD8
⫹
phenotype was the result of expansion of
a single aberrant clone by ex vivo analysis of V␤usage with a
panel of V␤-specific MAbs in the large expansions in subjects
A to D. Only in subject B was an expansion of a single V␤-
bearing subset seen (Fig. 2d, right panel), comprising 30% of
the tetramer-positive population at the first time point. This
was not seen in the other patients (for example, subject A [Fig.
2d, left panel]) and was not sustained in subject B, in whom the
proportion dropped to about 10% by week 14 (data not shown).
Thus, the observed phenotype of weak or absent cytokine
secretion upon stimulation appears to extend across popula-
tions of CD8
⫹
T lymphocytes targeting distinct epitopes and
using distinct TCRs.
Analysis of lytic function and perforin staining ex vivo. We
also had the opportunity with a single patient (subject A) to
address whether the cells identified at the peak of activation
during acute hepatitis were directly cytotoxic ex vivo. In this
situation, at the first time point, 3% of CD8
⫹
T lymphocytes
were NS3 1073 specific and were highly activated (92% CD38
high and 64% MHC class II high), as previously observed in
other patients (19) (Fig. 1b). Nevertheless at this time point,
no specific lysis of peptide-pulsed targets was observed in a 5-h
or extended assay for chromium release, even though the same
targets were readily lysed by a specific clone (data not shown).
Consistent with this finding, the cells were low in perforin
(1.5% positive) and high in CD27 (90% positive), a phenotype
which has been associated with a low lytic capacity and what
has been described as an “immature” or “early differentiation”
phenotype (2). Of eight responses tested for perforin staining,
all were ⱕ10% positive (mean, 3%; range, 0 to 10%) (data not
shown).
Analysis of function of CD8
ⴙ
T lymphocytes ex vivo in pa-
tients later after infection. We and others have previously
reported that levels of virus-specific CD8
⫹
lymphocytes (as-
sessed by tetramer) in patients identified outside the setting of
acute disease are generally very low, which renders analyses of
phenotype and function technically difficult without in vitro
manipulation (14, 19, 21). To further address the question of
whether the dysfunction of these cells is sustained long term,
we screened blood from another 56 HLA-A2-positive subjects
from time points outside the first 24 months of infection. This
was performed with tetramers containing the four different
HLA-A2-restricted peptides exactly as used in subjects A to D
(19–21). Where appropriate, we also tested subjects by using
HLA-B8 and -B7 tetramers (see Materials and Methods). We
obtained samples from a further three such individuals (two
PCR negative and one PCR positive) with expansions of HCV-
specific HLA-A2 tetramer-positive cells representing ⬎0.1%
of CD8
⫹
lymphocytes in whom intracellular cytokine staining
could be assessed after stimulation.
Examples of such an analysis (subject E) are shown in Fig. 3a
and b. These show, respectively, a profound defect in synthe-
sis of both TNF-␣and IFN-␥after peptide stimulation
and a relative defect in staining for TNF-␣after maximal
(PMA-ionomycin) stimulation, compared to CMV and EBV
responses. Subject E had been PCR negative after IFN-␣treat-
ment 5 years previously, and thus the defect cannot be attrib-
uted to ongoing viremia. It was sustained over a period of 1
year, with the relatively large HCV-specific tetramer-positive
population (0.3% of CD8
⫹
lymphocytes) maintained in a qui-
escent state (low in CD38 and HLA class II; similar to the
EBV- and CMV-specific populations; data not shown).
Internal and group comparisons between HCV-specific and
control responses. The relative lack of cytokine secretory ca-
pacity in HCV-specific CD8
⫹
T lymphocytes was further em-
phasized by comparisons with EBV- and CMV-specific re-
sponses within and between individuals. In Fig. 4 (upper
panel), the synthesis of IFN-␥after PMA stimulation from
HCV-specific CD8
⫹
lymphocytes is compared with that from
FIG. 4. Overall comparisons of HCV function and control re-
sponses. (Upper panel) Internal comparison. Five HCV antibody-
positive subjects (all HCV PCR negative) for whom HCV and control
responses were available were tested simultaneously for IFN-␥staining
after PMA-ionomycin stimulation, and the proportion of tetramer-
positive cells was calculated as in Fig. 2a, upper panels. Each HCV
response was compared with the EBV and/or CMV response in the
same individual (a total of eight comparisons). (Lower panel) Group
comparison. Cytokine synthesis from HCV tetramer-positive popula-
tions in seven HCV antibody-positive subjects (five PCR negative)
were tested exactly as described above (left-hand group, marked HCV)
and compared with EBV and/or CMV responses within themselves
and in seven normal controls (right-hand group). CMV and EBV
responses from HCV antibody-positive subjects are shown by solid
circles, and those from control subjects are shown by open circles.
5556 GRUENER ET AL. J. VIROL.

EBV- and CMV-specific populations in all of those individuals
in whom both responses were present. This shows a marked
skewing of the responses in favor of cytokine release from the
non-HCV-specific CD8
⫹
populations (P⬍0.002). In some
individuals (as in subject A), more than one HCV-specific
response was present, and both showed a similar phenotype by
comparison with CMV or EBV.
In Fig. 4 (lower panel), the IFN-␥responses after PMA
stimulation are compared between HCV tetramer-positive
populations and non-HCV (EBV or CMV) populations in
seven HCV antibody-positive (dark dots) and seven control
subjects (clear dots). The overall level of responsiveness is
again lower at the population level (mean, 13.5% versus 60%;
P⬍0.0001). There was no significant difference between non-
HCV (EBV and/or CMV) responses in HCV antibody-positive
and antibody-negative subjects.
DISCUSSION
These data indicate that HCV-specific CD8
⫹
T lymphocytes
possess a distinct phenotype, which may be maintained well
after virus is cleared from blood. Large populations of HCV-
specific CD8
⫹
lymphocytes are induced early and are highly
activated, as judged by surface expression of appropriate
marker molecules (21) (Fig. 1b), but have various degrees of
impairment of antiviral functions, as assessed in vitro. These
include an impaired ability to respond to peptide and to mi-
togen and are most obvious at the level of secretion of antiviral
cytokines.
The essential differences in response dynamics or function
dictating clearance versus persistence of HCV were not re-
vealed in this study. Relative defects in cytokine secretion were
seen in those who had cleared virus in the long term, and levels
of intracellular perforin were universally low among HCV-
specific CD8
⫹
lymphocytes. It is possible that the essential
mechanisms that control HCV in the long term lie outside of
these conventional functions or indeed are displayed by some
other subset of immune mediators, as has been clearly dem-
onstrated in murine models (26). An alternative explanation is
that the circulating CD8
⫹
lymphocytes represent an unusual
subset that differs from the truly functional lymphocytes. While
this seems likely during chronic hepatitis infection, where com-
partmentalization in the liver has been observed (23), it seems
unlikely in situations in which virus has been cleared and no
hepatitis is detectable.
These populations differ from previously identified dysfunc-
tional CD8
⫹
lymphocytes in that the phenotype is sustained
(21) and may occur in the presence of adequate CD4
⫹
help
(33), and the surface phenotype of the lymphocytes is other-
wise normal (e.g., CD45RO high; data not shown) (22). The
stability of these populations, compared with the dysfunction
seen in lymphocytes undergoing exhaustion (10), indicates a
long-lasting influence on function induced by the virus in an
antigen-specific manner. It is possible that the inducing envi-
ronment or potentially some influence on antigen-presenting
cell function (15) is responsible. For example, in murine mod-
els, a similar sustained phenotype has been seen after vaccina-
tion in the presence of interleukin-10 (5). In this context, a
description of “stunted” as opposed to “stunned” may be more
appropriate. How this may then have an impact on the persis-
tence of HCV remains to be determined, especially in the
context of the balance with other mediators of immunity (6, 9).
Methods to reverse this defect or to induce new populations of
functional T lymphocytes should be explored as part of immu-
notherapeutic strategies and vaccine design.
ACKNOWLEDGMENTS
Norbert Gruener and Franziska Lechner contributed equally to this
paper.
Funding was obtained from the Wellcome Trust, the Wilhelm-Sand-
er-Stiftung and the European Union (5th Framework, HCVacc, grant
no. QLK2-CT-1999-00356), the Deutsche Forschungsgemein, the Aus-
tralian Red Cross, the National Institutes of Health, and the Doris
Duke Charitable Foundation.
We are grateful to Jane Collier and the staff of the hepatitis and
immunology clinics at the John Radcliffe Hospital for providing pa-
tient samples to screen; Mike Bunce for tissue typing; Philip Goulder,
Rod Dunbar, Richard Cornall, Victor Appay, and Andrew McMichael
for helpful discussions; and A. Morse for preparation of the manu-
script. We also thank Carmen Amsel, Barbara Becker, Jutta Do¨hr-
mann, and Marion Satzger for excellent technical assistance and per-
sonal encouragement.
REFERENCES
1. Altman, J., P. A. H. Moss, P. Goulder, D. Barouch, M. McHeyzer-Williams,
J. I. Bell, A. J. McMichael, and M. M. Davis. 1996. Direct visualization and
phenotypic analysis of virus-specific T lymphocytes in HIV-infected individ-
uals. Science 274:94–96.
2. Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King,
G. S. Ogg, H. M. Spiegel, C. Conlon, C. A. Spina, D. V. Havlir, D. D.
Richman, A. Waters, P. Easterbrook, A. J. McMichael, and S. L. Rowland-
Jones. 2000. HIV-specific CD8(⫹) T cells produce antiviral cytokines but are
impaired in lytic function. J. Exp. Med. 192:63–75.
3. Callan, M., L. Tan, N. Annels, G. Ogg, J. Wilson, C. O’Callaghan, N. Steven,
A. McMichael, and A. Rickinson. 1998. Direct visualisation of antigen spe-
cific CD8⫹T cells during the primary immune response to EBV in vivo. J.
Exp. Med. 187:1395–1402.
4. Cerny, A., J. McHutchinson, C. Pasquellini, M. Brown, M. Brothers, B.
Grabschied, P. Fowler, M. Houghton, and F. Chisari. 1995. CTL response to
HCV-derived peptides containing the HLA-A2.1 binding motif. J. Clin.
Investig. 95:521–530.
5. Chun, S., M. Daheshia, S. Lee, S. K. Eo, and B. T. Rouse. 1999. Distribution
fate and mechanism of immune modulation following mucosal immunisa-
tion. J. Immunol. 163:2393–2402.
6. Ciurea, A., P. Klenerman, L. Hunziker, E. Horvath, B. M. Senn, A. F.
Ochsenbein, H. Hengartner, and R. M. Zinkernagel. 2000. Viral persistence
in vivo through selection of neutralizing antibody-escape variants. Proc. Natl.
Acad. Sci. USA 97:2749–2754.
7. Cramp, M. E., S. Rossol, S. Chokshi, P. Carucci, R. Williams, and N. V.
Naoumov. 2000. Hepatitis C virus-specific T-cell reactivity during interferon
and ribavirin treatment in chronic hepatitis C. Gastroenterology 118:346–
355.
8. Diepolder, H. M., R. Zachoval, R. M. Hoffmann, E. A. Wierenga, T. San-
tantonio, M. C. Jung, D. Eichenlaub, and G. R. Pape. 1995. Possible mech-
anism involving T-lymphocyte response to non-structural protein 3 in viral
clearance in acute hepatitis C virus infection. Lancet 346:1006–1007.
9. Farci, P., A. Shimoda, A. Coiana, G. Diaz, G. Peddis, J. C. Melpolder, A.
Strazzera, D. Y. Chien, S. J. Munoz, A. Balestrieri, R. H. Purcell, and H. J.
Alter. 2000. The outcome of acute hepatitis C predicted by the evolution of
the viral quasispecies. Science 288:339–344.
10. Gallimore, A., A. Glitheroe, A. Godkin, A. Tissot, H. Hengartner, T. Elliott,
and R. Zinkernagel. 1998. Induction and exhaustion of LCMV-specific CTL
visualised using soluble tetrameric MHC-peptide complexes. J. Exp. Med.
187:1383–1393.
11. Gerlach, J., H. Diepolder, M.-C. Jung, N. Gruener, W. Schraut, R. Zachoval,
R. Hoffman, C. Schirren, T. Santantonio, and G. Pape. 1999. Recurrence of
HCV after loss of virus specific CD4⫹T cell response in acute hepatitis C.
Gastroenterology 117:933–941.
12. Gillespie, G. M. A., M. R. Wills, V. Appay, C. O’Callaghan, M. Murphy, N.
Smith, P. Sissons, S. Rowland-Jones, J. I. Bell, and P. A. H. Moss. 2000.
Functional heterogeneity and high frequencies of cytomegalovirus-specific
CD8
⫹
T lymphocytes in healthy seropositive donors. J. Virol. 74:8140–8150.
13. Guidotti, L., R. Rochford, J. Chung, M. Shapiro, R. Purcell, and F. Chisari.
1999. Viral clearance without destruction of infected cells during acute HBV
infection. Science 284:825–829.
14. He, X.-S., B. Rehermann, F. Lopez-Labrador, J. Boisvert, R. Cheung, J.
Mumm, H. Wedermeyer, M. Berenguer, T. Wright, and M. Davis. 1999.
VOL. 75, 2001 CYTOKINE RELEASE BY HCV-SPECIFIC CD8
⫹
T LYMPHOCYTES 5557

Quantitative analysis of HCV-specific CD8⫹T cells in peripheral blood and
liver using peptide-MHC tetramers. Proc. Natl. Acad. Sci. USA 96:5692–
5697.
15. Hiasa, Y., N. Horiike, S. M. Akbar, I. Saito, T. Miyamura, Y. Matsuura, and
M. Onji. 1998. Low stimulatory capacity of lymphoid dendritic cells express-
ing hepatitis C proteins. Biochem. Biophys. Res. Commun. 249:90–95.
16. Klenerman, P., F. Lechner, M. Kantzanou, A. Ciurea, H. Hengartner, and R.
Zinkernagel. 2000. Viral escape and the failure of cellular immune re-
sponses. Science 289:2003a.
17. Koziel, M. J., D. Dudley, N. Afdhal, A. Grakoui, C. M. Rice, Q. L. Choo, M.
Houghton, and B. D. Walker. 1995. HLA class I-restricted cytotoxic T lym-
phocytes specific for hepatitis C virus. Identification of multiple epitopes and
characterization of patterns of cytokine release. J. Clin. Investig. 96:2311–
2321.
18. Lalvani, A., R. Brookes, S. Hambleton, W. J. Britton, A. V. Hill, and A. J.
McMichael. 1997. Rapid effector function in CD8⫹memory T cells. J. Exp.
Med. 186:859–865.
19. Lechner, F., N. H. Gruener, S. Urbani, J. Uggeri, T. Santantonio, A. R.
Kammer, A. Cerny, R. Phillips, C. Ferrari, G. R. Pape, and P. Klenerman.
2000. CD8⫹T lymphocyte responses are induced during acute hepatitis C
virus infection but are not sustained. Eur. J. Immunol. 30:2479–2487.
20. Lechner, F., J. Sullivan, H. Spiegel, D. Nixon, B. Ferrari, A. Davis, B.
Borkowski, H. Pollack, E. Barnes, G. Dusheiko, and P. Klenerman. 2000.
Why do cytotoxic T lymphocytes fail to eliminate HCV? Lessons from
studies using MHC class I tetramers. Phil. Trans. R. Soc. Lond. B. 355:1085–
1092.
21. Lechner, F., D. K. Wong, P. R. Dunbar, R. Chapman, R. T. Chung, P.
Dohrenwend, G. Robbins, R. Phillips, P. Klenerman, and B. D. Walker.
2000. Analysis of successful immune responses in persons infected with
hepatitis C virus. J. Exp. Med. 191:1499–1512.
22. Lee, P. P., C. Yee, P. A. Savage, L. Fong, D. Brockstedt, J. S. Weber, D.
Johnson, S. Swetter, J. Thompson, P. D. Greenberg, M. Roederer, and M. M.
Davis. 1999. Characterization of circulating T cells specific for tumor-asso-
ciated antigens. Nat. Med. 5:677–685.
23. Minutello, M., P. Pileri, D. Unutmaz, S. Censini, G. Kuo, M. Houghton, M.
Brunetto, F. Bonino, and S. Abrignani. 1993. Compartmentalization of T
lymphocytes to the site of disease: intrahepatic T cells specific for the NS4
protein of HCV in patients with chronic hepatitis C. J. Exp. Med. 178:17–25.
24. Missale, G., R. Bertoni, V. Lamonaca, A. Valli, M. Massari, C. Mori, M.
Rumi, M. Houghton, F. Fiaccadori, and C. Ferrari. 1996. Different clinical
behaviours of acute HCV infection are associated with different vigor of the
anti-viral T cell response. J. Clin. Investig. 98:706–714.
25. Naoumov, N. V. 1999. Hepatitis C virus-specific CD4(⫹) T cells: do they help
or damage? Gastroenterology 117:1012–1014.
26. Planz, O., S. Ehl, E. Furrer, E. Horvath, M. Brundler, H. Hengartner, and
R. Zinkernagel. 1997. A critical role for neutralising antibody-producing B
cells, CD4⫹cells and interferons in persistent infections of mice with
LCMV: implications for adoptive immunotherapy of virus carriers. Proc.
Natl. Acad. Sci. USA 94:6874–6879.
27. Rehermann, B., K. Chang, J. McHutchinson, R. Kokka, M. Houghton, and
F. Chisari. 1996. Quantitative analysis of the peripheral blood CTL response
in patients with chronic HCV infection. J. Clin. Investig. 98:1432–1440.
28. Rosen, H. R., D. J. Hinrichs, D. R. Gretch, M. J. Koziel, S. Chou, M.
Houghton, J. Rabkin, C. L. Corless, and H. G. Bouwer. 1999. Association of
multispecific CD4(⫹) response to hepatitis C and severity of recurrence after
liver transplantation. Gastroenterology 117:926–932.
29. Takaki, A., M. Wiese, G. Maertens, E. Depla, U. Seifert, A. Liebetrau, J. L.
Miller, M. P. Manns, and B. Rehermann. 2000. Cellular immune responses
persist and humoral responses decrease two decades after HCV infection.
Nat. Med. 6:578–582.
30. Thursz, M., R. Yallop, R. Goldin, C. Trepo, and H. C. Thomas. 1999.
Influence of MHC class II genotype on outcome of infection with hepatitis
C virus. The HENCORE group. Hepatitis C European Network for Coop-
erative Research. Lancet 354:2119–2124.
31. Whelan, J. A., P. R. Dunbar, D. A. Price, M. A. Purbhoo, F. Lechner, G. S.
Ogg, G. Griffiths, R. E. Phillips, V. Cerundolo, and A. K. Sewell. 1999.
Specificity of CTL interactions with peptide-MHC class I tetrameric com-
plexes is temperature dependent. J. Immunol. 163:4342–4348.
32. Wong, D., D. Dudley, N. Afdhal, J. Dienstag, C. Rice, L. Wang, M. Houghton,
B. Walker, and M. Koziel. 1998. Liver derived CTL in HCV infection:
breadth and specificity of responses in a cohort of patients with chronic
infection. J. Immunol. 160:1479–1488.
33. Zajac, A., J. Blattman, K. Murali-Krishna, D. Sourdive, M. Suresh, J.
Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence of
activated T cells without effector function. J. Exp. Med. 188:2205–2213.
5558 GRUENER ET AL. J. VIROL.