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* Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;
Stanford University School of Medicine, Stanford, CA 94305;
Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production of
HCV-tetramer+ cells, but not influenza-virus-specific T
cells, were defective in chronically infected patients and could not be
restored by in vitro stimulation with peptide and IL-2. At least three
distinct phenotypes of HCV-specific CD8+ T cells were
identified and associated with certain functional characteristics. In
addition, impairment of proliferative, cytokine, and cytotoxic effector
functions of tetramer+ T cells in viremic patients was
associated with weak ex vivo HCV-specific CD4+ T cell
responses. Thus, the defective functions of HCV-specific
CD8+ T cells might contribute to viral persistence in
chronically infected patients, and knowledge on their reversibility may
facilitate the development of immunotherapeutic
vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of
cases with acute hepatitis, 70% with chronic hepatitis, 40% with
end-stage cirrhosis, 60% with hepatocellular carcinoma, and 15–30%
of liver transplantations (1). In acute HCV infection, an early HCV-specific T cell response is associated with viral clearance and recovery (2, 3, 4, 5, 6, 7, 8), and HCV-specific CTLs persist at least two decades after recovery from hepatitis C (9). In contrast, in chronically infected individuals, cellular immune responses appear to be too weak to eliminate HCV (9). Whether this weakness is due to a low frequency of HCV-specific T cells or to an impairment of their effector functions is not known.
Detailed analysis of the functional properties of HCV-specific
CD8+ T cells in chronically infected patients had
been hampered by the low frequency of such cells and the requirement
for extensive in vitro expansion. Quantification of HCV-specific
CD8+ T cells was initially based on limiting
dilution analysis and thus was dependent on the frequency of specific
cells, their in vitro expansion potential, and their ability to lyse
appropriate targets (10). Subsequently, the use of the
IFN- ELISPOT for direct ex vivo analysis demonstrated that the
frequency of virus-specific circulating CD8+ T
cells was significantly underestimated by limiting dilution analysis
(11). However, even the ELISPOT assay might not reflect
the true number of virus-specific T cells because quantification of
cells depends on cytokine production in this assay. The introduction of
MHC class I-peptide tetramers that bind to the TCRs of Ag-specific
cells now allows enumeration of specific CD8+ T
cells regardless of their effector function (12) and
provides additional information on their phenotype if combined with
staining for cell surface markers and intracellular cytokines.
Virus-specific CD8+ T cells have been
investigated with tetramers in several diseases including HIV
(12, 13), EBV (14), and CMV infection
(13), and we and others have reported the use of tetramers
in studies of HCV (6, 7, 15, 16).
In this study, we used four HLA-A2.1 tetramers specific for HCV core
and NS3 epitopes, respectively, in combination with proliferation,
IFN- ELISPOT, and cytotoxicity assays to investigate the frequency
of HCV-specific T cells and the effector functions of these cells
directly ex vivo and in short-term-cultured (day 7) T cell lines. We
found a higher frequency of circulating tetramer+
T cells in chronic than in recovered patients, but
tetramer+ cells of chronic patients were
functionally impaired, i.e., did not expand after Ag stimulation, did
not produce IFN- in response to HCV peptides, and displayed less
HCV-specific cytotoxicity. These impaired functions were more
pronounced in the presence of weak ex vivo HCV-specific
CD4+ T cell responses and were associated with a
distinct CD45RO-CD27+
phenotype. Thus, the defective functions of HCV-specific
CD8+ T cells might contribute to viral
persistence in chronically infected patients, and knowledge on their
reversibility may facilitate the development of immunotherapeutic
vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty chronically infected, untreated HCV-RNA-positive patients (genotypes 1 or 2b) with alanine aminotransferase levels within 3-fold of the upper limit of normal were compared with 12 recovered patients who had been HCV-RNA negative for up to 12 years. Eight healthy blood donors without a history of HCV infection served as controls. All subjects were HLA-A2 positive, had been followed in the Department of Transfusion Medicine Clinical Center and the Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases (National Institutes of Health, Bethesda, MD), and gave informed consent to this study, which was approved by the institutional review board of National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Anti-HCV testing, qualitative PCR for HCV RNA, and genotyping were performed as previously described (17).
Peptides and Abs
Four HLA-A2-restricted HCV peptides (Core-35
YLLPRRGPRL, Core-132 DLMGYIPLV, NS3-1073 CVNGVCWTV, and NS3-1406
KLVALGINAV) (9, 18, 19), the influenza A virus matrix
peptide GILGFVFTL (20) (Research Genetics, Huntsville,
AL), and the following mAbs were used: anti-CD4-FITC,
anti-CD13-FITC, anti-CD19-FITC, anti-CD27-FITC,
anti-CD28-FITC (Caltag Laboratories, Burlingame, CA),
anti-CD8-PerCP, anti-CD8-FITC (BD Biosciences, San Jose, CA),
anti-CD45RA-FITC, anti-CD45RO-FITC, anti-CD152-CyChrome,
anti-perforin-PE, anti-IFN--FITC (BD PharMingen, San Diego,
CA), anti-CD69-FITC, anti-CD94-FITC (Ancell, Bayport, MN), and
anti-perforin-FITC (Research Diagnostics, Flanders, NJ).
Generation of T cell lines
PBMCs were separated on Ficoll-Histopaque (Sigma-Aldrich, St.
Louis, MO) as described (9). PBMCs were stimulated in
96-well round-bottom plates (0.4 x 106 per
well) with 10 µg/ml peptide in RPMI 1640, 10% heat-inactivated human
serum (blood group AB), L-glutamine (2 mM), penicillin (100
U/ml), and streptomycin (100 µg/ml). On day 4, 100 µl of RPMI 1640
with 10% AB serum and 10 U/ml rIL-2 was added. On day 7, the number of
tetramer-specific CD8+ T cells was determined by
flow cytometry, cytotoxicity by CTL assay, and IFN- production by
intracellular cytokine staining. For an additional CTL assay after 3 wk
of culture, T cell lines were maintained as previously described
(9).
Tetramer staining of PBMCs and T cell lines
Peptide-MHC tetramers were prepared by Dr. Mark Davis (Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA), the National Institute of Allergy and Infectious Diseases Tetramer Facility, and Otsuka Pharmaceuticals (Tokushima, Japan). PBMCs and T cell lines were stained as described (15). The detection limit was 0.01% of CD8+ cells for the NS3-1073 tetramer and the NS3-1406 tetramer, 0.03% of CD8+ cells for the Core-35 tetramer, and 0.02% of CD8+ cells for the Core-132 tetramer. The detection limit was determined as the background signal plus three SDs after staining PBMCs from HLA-A2+, HCV-negative blood donors (n = 6) and from HLA-A2-negative patients with chronic hepatitis C (n = 5).
Because HCVNS3-1073-specific T cells could
potentially have been induced by exposure to a previously described
cross-reactive influenza A virus (A/PR8/34) neuraminidase peptide of
similar sequence (21), we tested PBMCs from patients with
detectable
HCVNS3-1073-tetramer+ cells
(patients chronic hepatitis C (Chr)-14, Chr-16, Chr-2, and Chr-15, as
shown in Fig. 1
) for recognition of the
cross-reactive peptide in an IFN- ELISPOT assay. None of the
patients displayed any immune responses against the influenza A virus
neuraminidase peptide (data not shown), indicating that the responses
were HCV specific.
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Ten million PBMCs were stained with 1 µM CFSE (Molecular Probes, Eugene, OR) (22) in 1 ml of PBS for 10 min at 37°C. The reaction was stopped with FCS and the cells were washed three times in PBS. CFSE-labeled cells were then stimulated in a 7-day culture with 10 µg/ml peptide. On day 7, cells were stained with tetramers and anti-CD8 and were analyzed by flow cytometry.
Cytotoxicity assay
HCV-specific cytotoxicity was determined by standard 51Cr release assay (9) against 3 x 103 peptide-pulsed C1R-A2.1 cells, kindly provided by Dr. J. Berzofsky (23) in the presence of a 40-fold excess of unlabeled K562. Unstimulated PBMCs or day 7 or day 21 CTL lines were added at varying E:T ratios. Spontaneous release was <7% of maximum release in all 6-h CTL assays and <15% in all 12-h CTL assays.
Intracellular cytokine staining and ELISPOT assays
PBMCs (2 x 106 cells/ml) were
stained with tetramers and thereafter stimulated with or without
peptide (10 µg/ml) for 2 h at 37°C and an additional 4 h
in the presence of brefeldin A, followed by a 15-min incubation at room
temperature in 0.02% EDTA. Intracellular cytokine staining for IFN-
(0.25 µg of anti-IFN-/106 cells, BD
PharMingen) was performed using the Cytofix/CytoPerm kit (BD
PharMingen) according to the manufacturer’s instructions. Cells were
washed twice, and 500,000 events in the lymphogate were collected by
flow cytometry (FACSCalibur; BD Biosciences) and analyzed with
CellQuest software (BD Biosciences). ELISPOT assays for IFN- and
IL-5 were performed exactly as previously described (9).
Spots were counted with an automated ELISPOT reader (KS Elispot; Zeiss,
Thornwood, NY) and evaluated based on size, shape, contrast, and
density. Typically, spots were irregularly shaped (because lymphocytes
move during the 36-h incubation period), displayed a fuzzy border, and
had a darker and denser center. Based on extensive control experiments,
we consistently used 1000 µm2 as a cutoff to
exclude very small spots, which were likely due to streptavidin
precipitates and other artifacts.
Statistical analysis
Kruskall-Wallis’ ANOVA, Student’s t tests, and linear regression analysis using Spearman’s correlation coefficient were performed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unstimulated PBMCs from 20 patients with chronic
hepatitis C and 12 long-term recovered patients (Table I) were screened for the presence and
frequency of HCVCore-35,
HCVCore-132, HCVNS3-1073,
and HCVNS3-1406 tetramer+
cells. In contrast with higher frequencies of EBV- and CMV-specific T
cells that have been reported in patients infected with those viruses
(24, 25, 26), the number of HCV-specific T cells was very low
in the blood of patients with chronic hepatitis C, with a mean
frequency of only 0.05 ± 0.18% circulating
CD8+ T cells (range, 0.0–1.2%; Fig. 1
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), consistent with a further reduction of Ag-specific cells after
clearance of the virus. In fact, when all experiments with individual
tetramers were considered, HCV-specific tetramer+
cells were detected in 6 of 44 (14%) experiments with PBMCs from
recovered patients as compared with 25 of 78 (32%) experiments
performed with unstimulated PBMCs from chronically infected patients
(p = 0.016, Kruskall-Wallis test). In addition,
none of the recovered patients, but 7 of 20 chronically infected
patients, tested positive with multiple tetramers
(p = 0.02). This difference between recovered and chronically infected patients remained significant even when the two data points with very high numbers of tetramer+ cells (HCVNS3-1073-specific cells of patient Chr-14 and HCVNS3-1406-specific cells of patient Chr-16) were excluded. Omission of these two data points resulted in 6 of 44 (14%) positive assays in recovered patients as compared with 23 of 76 (30%) positive assays in chronically infected patients (p = 0.041, Kruskall-Wallis test).
Similarly, omission of these two data points did not change the finding that 7 of 20 chronically infected patients but none of the recovered patients tested positive with multiple tetramers (p = 0.02). Complete omission of patients Chr-14 and Chr-16, who tested positive for three and four tetramers, respectively, resulted in 5 of 18 chronically infected as compared with none of the recovered patients testing positive with multiple tetramers (p = 0.049).
Effector functions of tetramer+ cells
Proliferation. To investigate the proliferative
capacity of HCV-specific CD8+ T cells at the
single-cell level, we stained PBMCs with the fluorescent dye CFSE
(22) and determined the CFSE content of
tetramer+ cells after 7 days of in vitro
stimulation with HCV epitope peptides. For patients Chr-16, Chr-17, and
Chr-19, the intensity of the CFSE signal remained high after 7 days of
in vitro stimulation, as shown by the tetramer+
cells in the upper right quadrants of the dot plots (Fig. 2A), indicating that the cells
were viable but failed to expand. Similarly, approximately two-thirds
of the HCVNS3-1073-specific,
tetramer+ cells of patient Chr-14 retained their
CFSE signal, indicating that the majority of
tetramer+ cells did not divide (Fig. 2A).
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A, cells in the upper left
quadrants), indicating that they had expanded during 7 days of
culture (Fig. 2A). This high proliferative potential of
tetramer+ cells of recovered patients was peptide
specific, because it was only observed in response to the specific
peptide, not in response to an unrelated peptide (Fig. 2B).
Overall, 44 tetramer stainings were performed with cells of recovered
patients and 67 stainings were performed with cells of chronic
patients, demonstrating that the proliferative capacity of HCV-specific
CD8+ T cells was significantly stronger in
recovered than in chronically infected patients. HCV
tetramer+ cells were detectable in 6 of 44 direct
ex vivo experiments (14%) with unstimulated PBMCs and in 26 of 44 day
7 T cell cultures (59%) of recovered patients (p <
0.0001, Kruskall-Wallis test; Fig. 2C). Thus, low numbers of
HCV-specific CD8+ T cells (<0.01% of
CD8+ T cells, the detection limit of the tetramer
staining) expanded significantly and, after 7 days of specific
stimulation, represented up to 12% of all CD8+ T
cells in the culture (data not shown). The expansion rate was greatest
for HCVNS3-1073-specific T cells (2.4 ±
4.4% in the day 7 culture), followed by
HCVCore-132 and
HCVNS3-1406-specific T cells (0.25 ± 0.25%
and 0.24 ± 0.29%, respectively), and was lowest for
HCVCore-35-specific T cells (0.03 ± 0.05%)
(data not shown).
To demonstrate that non-HCV-specific T cells of chronically infected
patients proliferated normally, we analyzed CD8+
T cells specific for the influenza A virus
epitopeMA-58 (20), an immunodominant
and highly conserved epitope. As demonstrated in Fig. 3, the proliferative capacity of
influenza A virusMA-58-specific
CD8+ T cells of patients with chronic HCV
infection was not impaired. In fact, CFSE-labeled
influenzaMA-58-specific
tetramer+ cells of these patients proliferated to
the same extent as tetramer+ cells of recovered
patients after 7 days of in vitro stimulation.
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Cytotoxicity. To analyze effector functions of HCV-specific T cells, PBMCs of recovered and chronic patients were subjected to one to three rounds of weekly HCV peptide restimulation and were tested for peptide-specific cytotoxicity in standard 51Cr release assays.
Although PBMCs of recovered patients did not display significant
peptide-specific cytotoxicity when tested directly ex vivo (data not
shown), a single restimulation with Ag gave rise to daughter cells with
significant peptide-specific cytotoxic effector function (Fig. 4). When HCV peptide-specific T cell
lines were established from PBMCs of 8 recovered and 20 chronically
infected patients, significant cytotoxic activity was more frequently
detected in T cell lines derived from recovered individuals than in
those derived from chronic patients (Fig. 4A). To examine
whether this difference in the overall cytotoxicity of HCV
peptide-stimulated T cell lines between both patient groups was due to
different levels of cytotoxicity at the single-cell level, we
determined the number of tetramer+ T cells in a
subgroup of all cultures and calculated the true effector (i.e.,
tetramer+ cell) to target cell ratio reflecting
the percent specific cytotoxicity at the single
tetramer+ T cell level. Significant cytotoxicity
was still detectable at a tetramer+ cell:target
ratio as low as 0.05:1 (Fig. 4B), which is comparable to
that of T cell clones (13). In both recovered and
chronically infected patients, the cytotoxic activities of the T cell
lines correlated well with the number of
tetramer+ cells (r = 0.80 and
0.78, respectively; Fig. 4B). Cytotoxicity per
tetramer+ cell tended to be higher in recovered
than in chronically infected patients, but the difference was not
significant.
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IFN- production.
To investigate ex vivo IFN- production of peptide-specific
CD8+ T cells, we performed ELISPOT and
intracellular cytokine staining assays. As previously reported by other
investigators (27), we found that ELISPOT assay and
intracellular cytokine staining detected similar numbers of
IFN--producing cells (data not shown). As shown in Fig. 5A, significantly fewer
HCV-tetramer+ T cells than influenza A
virusMA58-tetramer+ T cells
produced IFN- in chronically infected patients when ex vivo analysis
was performed. Specifically, in 11 of 14 (79%) chronically infected
patients, HCV-tetramer+ cells produced IFN- in
response to their specific antigenic peptide. In recovered patients,
this analysis could not be performed for most cases because the
frequency of HCV-tetramer+ cells in the blood was
below the detection level. However, in three recovered patients with
detectable tetramer+ cells and ELISPOT results,
40–100% of HCV-tetramer+ cells produced
IFN-.
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production in most cases (Fig. 5B). Phenotypic and functional analysis of HCV-specific CD8+ T cells at the single-cell level
We then performed a detailed analysis of the phenotype of HCV-specific cells in patients with a sufficiently high quantity of tetramer+ T cells. At least three different phenotypes of HCV-specific, tetramer+ CD8+ T cells were identified.
Table II focuses on
tetramer+ T cell populations of patients Rec-2,
Chr-14, Chr-16, and Chr-8 to demonstrate the phenotypic and functional
characteristics of HCV-specific cells. In these patients, the frequency
of tetramer+ cells and the amount of blood
available allowed a larger number of assays. In addition, the frequency
of these tetramer+ cells in the peripheral blood
was relatively constant during 20–39 mo of follow-up, with <3-fold
changes (data not shown), and thus represented stable responses.
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and data not shown).
Direct ex vivo phenotyping characterized these cells as
CD45RO+RA- memory cells
(Fig. 6) with characteristically high
expression level of the costimulatory molecules CD27 and CD28 (Table II). Low levels of HLA-DR were consistent with a resting phenotype,
indicating viral clearance and lack of recent Ag contact in vivo.
Although these cells contained low levels of prestored perforin and
were not cytotoxic in a direct ex vivo cytotoxicity assay (Table II),
they displayed a rapid recall response upon in vitro reexposure to the
specific HCV peptide. This recall response was evidenced by multiple
effector functions, including proliferation (Fig. 2A),
IFN- production (Fig. 6), and generation of daughter cells, which
were highly cytotoxic with 17 and 53% specific cytotoxicity at E:T
ratios of 2:1 and 30:1, respectively (data not shown).
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). Despite CD45RO expression, however, these cells should be
considered fully mature effector cells because CD28 as well as CD27
were down-regulated as also described for fully differentiated effector
cells in CMV infection. The observed high level of HLA-DR expression
(Fig. 6) was consistent with this fully differentiated effector cell
type (28) and may indicate recent Ag contact and
activation in vivo. Moreover, these cells contained large amounts of
perforin prestored in intracytoplasmic vesicles (Table II) and were
able to exert HCV-specific cytotoxicity directly ex vivo without the
need of additional restimulation (Table II). Importantly, T cells with
down-regulated CD28 expression have also recently been demonstrated in
the liver of patients with chronic hepatitis C (29). Our
data suggest that they may contribute to liver injury based on their
cytotoxic effector functions, but may be unable to completely
eradicate the virus because proliferation (Fig. 2A) and
IFN- production were suboptimal (Fig. 6 and Table II).
In contrast with chronically infected patient Chr-14, patient Chr-16
displayed tetramer+ cells of a different
phenotype with significantly impaired function. These cells were
CD45RO-RA+ (Fig. 6) and
did not express CD28. Despite down-regulation of CD28, however, 40%
of the tetramer+ cells were
CD27+ (Table II), indicating that these cells had
not yet reached the same maturation stage as the effector cells of
patient Chr-14 (30, 31). Interestingly, this particular
CD45RA+CD27+ phenotype was
not only found for HCVNS3-1073-specific T cells
of patient Chr-16, but also for a second NS3 epitope in the same
patient (Table II), for two HCVCore epitopes in
patient Chr-8 (Table II), and for the HCVCore-132
epitope in patient Chr-10 (data not shown). Again, all cells tested
negative for all analyzed effector functions, including direct ex vivo
cytotoxicity, peptide-specific IFN- production, and in vitro
proliferation.
Importantly, impairment of function was not associated with significant
expression of CD152 (CTLA4; Fig. 6 and Table II), a receptor reported
to regulate CD8+ T cell functions (32, 33). Similarly, Abs against CD94 did neither restore
HCV-specific cytotoxicity nor IFN- production (data not shown),
indicating that signaling through these two inhibitory receptors was
not responsible for the impaired function of
CD8+ T cells. We also excluded the presence of
viral escape mutations as a potential cause for the impaired T cell
function of patient Chr-16 by sequencing the epitope coding regions
within the virus (data not shown).
Thus, three different phenotypes of HCV-specific T cells were identified. First, memory T cells were not activated and did not display direct ex vivo effector function, a finding that supports the hypothesis that HCV is completely cleared in recovered individuals. Second, in chronically infected patients, two different phenotypes of tetramer+ cells were identified and the CD45RO-RA+CD27+ phenotype, detected for four different epitope specificities, was associated with the complete absence of all analyzed ex vivo and in vitro effector functions.
Ex vivo analysis of HCV-specific CD4+ T cell responses in chronically infected patients
Because it has been suggested in other studies that
CD28-CD27+ cells might be
arrested at an early stage of maturation due to a lack of
CD4+ T cell help (34), we
investigated HCV-specific CD4+ T cell responses
of all patients enrolled in this study by direct ex vivo IFN-
ELISPOT analysis. Overall, the number of circulating T cells that
produced IFN- in response to the HCV proteins core, NS3, NS4,
NS5A, and NS5B was significantly higher for recovered than for
chronically infected patients (Table III). IFN- ELISPOT assays with
separated CD4+ and CD8+ T
cells identified these responding cells as CD4+
Th cells (data not shown). In addition, the vigor of HCV-specific
CD4+ Th1 responses in patients Rec-2, Chr-14,
Chr-16, and Chr-8 correlated strongly with the observed effector
functions of their HCV-specific CD8+ T cells. For
example, a high frequency of Th cells that recognized five different
HCV proteins was found in patient Rec-2. In contrast, patients Chr-16
and Chr-8, who showed impaired CD8+ T cell
functions, displayed significantly lower frequencies of HCV-specific Th
cells, and the responses were targeted against fewer proteins (Table III and Fig. 7). Furthermore, patient
Chr-14, who maintained some degree of CD8+ T cell
function (Fig. 2 and Table II), was also the only chronically infected
patient with a significant HCV-specific IFN- response against any
HCV protein (Fig. 7), indicating that detectable effector function of
HCV-specific CD8+ or CD4+ T
cells is a less frequent event in chronic hepatitis C.
IL-5production in response to the same HCV proteins was
evaluated as a marker for HCV-specific CD4+ Th2
responses. Importantly, IL-5 responses were even weaker than IFN-
responses (Table IV), indicating that the
absence of HCV-specific CD4+ Th1 responses in the
blood of chronically infected patients was not due to a Th2 switch.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first finding of our study was that the number of HCV-specific tetramer+ CD8+ T cells was extremely small in both recovered and chronically infected patients. This is in sharp contrast with infections with other pathogens such as influenza A virus, EBV, and CMV, for which the frequency of epitope-specific T cells has been reported to be as high as 30–50% CD8+ T cells (24, 25, 26, 36). Thus, in contrast to those viruses, HCV appears to be capable of efficiently turning down the CD8+ T cell response, which may also explain the clinically asymptomatic onset of HCV infection and the mild degree of liver injury in the majority of patients (37, 38, 39).
However, the low frequency of HCV-specific T cells did not solely
explain the weak cellular immune response of chronically infected
patients. Even when we did find circulating, HCV-specific
tetramer+ T cells in the blood of chronically
infected patients, their effector function appeared to be impaired.
When compared with HCV-tetramer+ T cells of
recovered patients and to influenza A virus-specific cells,
HCV-tetramer+ T cells of chronic patients
displayed impaired proliferative capacity and IFN- production upon
in vitro stimulation with the specific HCV peptide. Although
cytotoxicity was observed in some instances at the single-cell level,
impaired proliferation of tetramer+ T cells
interfered with an effective amplification of cytotoxic effector
functions. As a result of the impaired proliferative capacity, fewer
HCV-specific T cell lines from chronically infected patients displayed
cytotoxicity after in vitro culture than those from recovered
patients.
These data suggest that HCV-specific T cells may contribute to liver
injury in chronic infection, because cells with this specificity
(15, 40, 41) and phenotype (29) have been
detected in liver tissue and have been shown to exert some degree of
cytotoxic effector function (41). Nevertheless, they may
be unable to completely eradicate the virus because of their functional
defects, as indicated by impaired IFN- production (Figs. 5 and 6)
and in vitro proliferation (Fig. 2A). These data extend
previous reports on reduced TNF-
and IFN- production of
HCV-specific T cells, as compared with EBV- and CMV-specific T cells
(16). In addition, functionally defective
CD8+ T cells have also been described in other
chronic infections, for example with HIV (13, 35, 42), and
in malignancies (43). This indicates that our finding is
not unique for HCV infection, but rather it is an example of a more
general phenomenon in the context of persistent Ag.
The mechanisms responsible for these suboptimal functions of
HCV-specific T cells in chronically infected patients remain to be
elucidated. Several potential explanations have previously been
proposed. First, rapid T cell death, as reported for highly activated
effector T cells upon restimulation (44), does not appear
to be the case in our study, because HCV-specific,
tetramer+ T cells were still detectable 7 days
after in vitro stimulation, even though they did not proliferate (Fig. 2A). Second, we have excluded a significant expression of
inhibitory NK cell receptors such as CD94 and CD152 on the majority of
dysfunctional T cells by flow cytometry (Fig. 6), and in contrast to a
previous report on melanoma-specific T cells, Abs to CD94 did not
restore the function of tetramer+ T cells in
response to Ag in this study. Third, HCV variants with altered epitope
sequences have been described as mechanisms of viral escape from T cell
responses (45, 46). We have excluded viral escape mutation
as the cause for the impaired T cell phenotype of patient Chr-16 by
determining the epitope coding HCV sequence in PCR products and
molecular clones (data not shown). Although we cannot exclude the
possibility of viral escape in the other samples that we could not
sequence, it should be considered that the T cell response was
generally weak in our chronically infected patients. Even when escape
mutants were identified in chimpanzees experimentally inoculated with
HCV, T cells specific for wild-type sequences still have been expanded
from liver tissue (46, 47). Detection of functional
virus-specific CD8+ T cells in recovered
patients, although at low levels, and of wild-type-specific
CD8+ T cells in chronically infected patients
with escape mutants (45, 48), also indicates that
CD8+ T cells can be maintained and continue to
circulate in the blood despite the absence of their cognate Ags. Thus,
although viral escape mutants cannot be excluded in all patients of
this study, it is still informative that the effector function of the
majority of HCV-specific T cells in chronically infected patients was
impaired. Serum samples from the very early course of infection or the
infectious source would be required to evaluate the sequence of the
original infecting virus.
Fourth, induction of anergy by high Ag levels remains another possible explanation. In hepatitis B virus (HBV) infection, large amounts of HBe Ag are secreted into the blood and have been implicated in mediating neonatal T cell tolerance (49) and in altering the reactivity of HBe-specific CD8+ T cells (50). Although the concentration of free, circulating viral protein is several logs lower in chronic HCV than in HBV infection and although HCV Ags are not produced in a specific secreted form such as HBe Ag in HBV infection (51), very low concentrations of HCV core Ag have been implicated in a down-regulation of cellular immune responses (52, 53). In addition, the amount and effect of viral Ags as part of viral particles coated with low- and very low-density lipoproteins (54) remain unknown.
Finally, in this study, we report a phenotypic heterogeneity among
HCV-specific T cells that was associated with distinct patterns of
effector functions. Although the number of
tetramer+ patients was too small to assess the
significance of each phenotype for disease progression and control of
viral load, the observed phenotypic and functional characteristics were
in accordance with published reports on other infections. For example,
down-regulation of CD28 and CD27 was observed on HCV-specific effector
cells of patient Chr-14. In accordance with recent reports that CD28
and CD27 are irreversibly down-regulated on fully differentiated
effector cells (13, 28, 55),
tetramer+ cells of patient Chr-14 displayed a
fully differentiated effector cell type and were able to mediate direct
ex vivo cytotoxicity and, at least to some extent, to proliferate and
to produce IFN-.
In addition, we also describe an example of a more extreme phenotype
with completely impaired effector functions such as HCV-specific
proliferation, cytotoxicity, and IFN- production. Although CD28 was
down-regulated on tetramer+ cells of patient
Chr-16, expression of CD27, a molecule from the TNF receptor
superfamily that is expressed on thymic emigrants (30) and
irreversibly lost during differentiation (31), was
maintained. According to the literature,
CD28-CD27+ T cells have
not been observed in healthy individuals. Instead, they were found in
persistent viral infections (13, 28) and it has been
suggested that these cells are arrested in an immature state due to a
lack of CD4+ T cell help (34).
Indeed, unresponsive CD8+ T cells that were
noncytotoxic and did not produce IFN- have been described in animal
models of chronic lymphocytic choriomeningitis virus or SIV infection
under conditions of Th cell deficiency (56, 57). Although
CD4+ T cells were not required for the induction
of CD8+ T cell responses during acute lymphocytic
choriomeningitis virus infection (58, 59), they were
indispensable for the maintenance of virus-specific
CD8+ T cell responses that control chronic viral
infections (60). In accordance with these results, we have
found that various effector functions of HCV-specific
CD8+ T cells were impaired in chronically
infected patients with weak HCV-specific CD4+ T
cell responses. CD4+ T cell responses were weak
with regard to IFN- production, and this weakness was not due a Th1
to Th2 shift. In addition, proliferation of CD4+
T cells has also been described to be weak in chronically infected
patients (4, 9, 61)
Because clearance of HCV has been associated with a vigorous and maintained CD8+ T cell response (5, 6, 8) and because CD4+ T cells appear to be required to prevent recrudescence (62), we propose that HCV-specific CD4+ T cell help is required from the time point of acute HCV infection to maintain the proper functions of HCV-specific CD8+ T cells, the main antiviral effector cells. Although CD4+ T cell help could consist of direct activation of CD8+ T cells via cytokines (63), it is notable that these CD8+ T cell functions could not be restored by addition of exogenous IL-2 in the present study. Alternatively, CD4+ T cell help could be indirect via CD40-mediated activation of professional APCs (64). This mechanism might be relevant because of accumulating evidence of abnormal dendritic cell function in chronic HCV infection (65, 66). Thus, in the presence of a weak CD4+ T cell response, dendritic cells might not be sufficiently stimulated and, in turn, might not appropriately activate CD8+ T cells. Prospective analysis of CD4+ and CD8+ T cell function during the natural course of HCV infection is needed to address these important issues.
Finally, our findings may also have several therapeutic implications. Successful vaccination protocols may depend not only on the induction of HCV-specific CD8+ T cells, but also on the maintenance of their function in vivo. Indeed, it has been shown that stimulation of CD8+ T cells alone by peptide vaccines is not sufficient to clear the virus in chronic hepatitis B (67). Secondly, the heterogeneity of T cell responses in different patients suggests that it may be important to stratify patients into different treatment groups. Finally, enhancement of CTL activity is always like a double-edged sword, and the important question requiring further study is how the optimal number, phenotype, and effector functions of Ag-specific T cells can be achieved to mediate viral clearance without causing detrimental immunopathology.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule, Hannover 30623, Germany.
3 Address correspondence and reprint requests to Dr. Barbara Rehermann, Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive, Room 9B16, Bethesda, MD 20892. E-mail address: Rehermann{at}nih.gov
4 Abbreviations used in this paper: HCV, hepatitis C virus; Rec, recovered; Chr, chronic hepatitis C; HBV, hepatitis B virus.
Received for publication February 26, 2002. Accepted for publication July 18, 2002.
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therapy for chronic hepatitis C. Hepatology 28:1121.[Medline]
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E. J. Wherry and R. Ahmed Memory CD8 T-Cell Differentiation during Viral Infection J. Virol., June 1, 2004; 78(11): 5535 - 5545. [Full Text] [PDF]
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D. D. Anthony, N. L. Yonkers, A. B. Post, R. Asaad, F. P. Heinzel, M. M. Lederman, P. V. Lehmann, and H. Valdez Selective Impairments in Dendritic Cell-Associated Function Distinguish Hepatitis C Virus and HIV Infection J. Immunol., April 15, 2004; 172(8): 4907 - 4916. [Abstract] [Full Text] [PDF]
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M. Lucas, A. L. Vargas-Cuero, G. M. Lauer, E. Barnes, C. B. Willberg, N. Semmo, B. D. Walker, R. Phillips, and P. Klenerman Pervasive Influence of Hepatitis C Virus on the Phenotype of Antiviral CD8+ T Cells J. Immunol., February 1, 2004; 172(3): 1744 - 1753. [Abstract] [Full Text] [PDF]
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C. Rollier, E. Depla, J. A. R. Drexhage, E. J. Verschoor, B. E. Verstrepen, A. Fatmi, C. Brinster, A. Fournillier, J. A. Whelan, M. Whelan, et al. Control of Heterologous Hepatitis C Virus Infection in Chimpanzees Is Associated with the Quality of Vaccine-Induced Peripheral T-Helper Immune Response J. Virol., January 1, 2004; 78(1): 187 - 196. [Abstract] [Full Text] [PDF]
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D. Candotti, J. Temple, F. Sarkodie, and J.-P. Allain Frequent Recovery and Broad Genotype 2 Diversity Characterize Hepatitis C Virus Infection in Ghana, West Africa J. Virol., July 15, 2003; 77(14): 7914 - 7923. [Abstract] [Full Text] [PDF]
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N. H. Shoukry, A. Grakoui, M. Houghton, D. Y. Chien, J. Ghrayeb, K. A. Reimann, and C. M. Walker Memory CD8+ T Cells Are Required for Protection from Persistent Hepatitis C Virus Infection J. Exp. Med., June 16, 2003; 197(12): 1645 - 1655. [Abstract] [Full Text] [PDF]
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A. Dolganiuc, K. Kodys, A. Kopasz, C. Marshall, T. Do, L. Romics Jr., P. Mandrekar, M. Zapp, and G. Szabo Hepatitis C Virus Core and Nonstructural Protein 3 Proteins Induce Pro- and Anti-inflammatory Cytokines and Inhibit Dendritic Cell Differentiation J. Immunol., June 1, 2003; 170(11): 5615 - 5624. [Abstract] [Full Text] [PDF]
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 R. Thimme, J. Bukh, H. C. Spangenberg, S. Wieland, J. Pemberton, C. Steiger, S. Govindarajan, R. H. Purcell, and F. V. Chisari Inaugural Article: Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease PNAS, November 26, 2002; 99(24): 15661 - 15668. [Abstract] [Full Text] [PDF]
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) patients. The
percentage of tetramer+ cells within the
CD8+CD4-CD13-CD19-
lymphocyte population is shown. The dashed line indicates the detection
limit for each tetramer (background signal plus three SDs), the solid
line indicates the average frequency of tetramer+ cells.
The numbers represent the patient identification numbers.
