Full-Breadth Analysis of CD8+ T-Cell Responses in
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病菌学杂志 2005年第20期
Partners AIDS Research Center and Infectious Disease Division, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 02129
Howard Hughes Medical Institute, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 02129
Nuffield Department of Clinical Medicine, Peter Medawar Building, University of Oxford, United Kingdom
CNRS-BioMerieux, 69365 Lyon, France
Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 02114
Medizinische Universitaetsklinik, Berufsgenossenschaftliche Kliniken Bergmannsheil, 44789 Bochum, Germany
ABSTRACT
Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.
INTRODUCTION
Hepatitis C virus (HCV) infection affects 170 million people worldwide and represents a major public health problem in many countries (24). After acute infection the majority of individuals develop chronic disease, which may result in hepatic failure and liver cancer (34). Persons who clear HCV infection spontaneously usually display vigorous and multispecific cellular immune responses (8, 14, 26, 37), but the mechanisms by which the virus evades immune responses in humans developing chronic infection remain unclear. CD8+ T cells are critical in antiviral defense (35), playing a crucial role in a number of acute and persistent virus infections, such as influenza virus (12), human immunodeficiency virus (HIV) (16, 30, 33), Epstein-Barr virus (EBV) (5, 6, 36), and cytomegalovirus (15). In the chimpanzee model strong and multispecific CD8 T-cell responses have been associated with spontaneous control of HCV (8), and the emergence of escape mutations has been associated with the development of viral persistence (11, 38). Studies addressing this important question in humans are limited, often to responses restricted by a single allele, such as HLA A2 (18, 25, 37, 39), and the critical relationship between the breadth and magnitude of CD8+ T-cell responses in the acute phase of infection and disease outcome remains to be defined.
Once chronic HCV infection is established, therapy with alpha interferon (IFN-) and ribavirin leads to successful virological outcomes in about 50 to 75% of cases (13, 27), depending on viral genotype and pretreatment viral loads. Recent studies have suggested that therapy during the acute phase of infection is much more effective, with up to 95% of patients becoming virus-free after a relatively short course of alpha interferon monotherapy (19, 29). It has been suggested that cellular immune responses, which are strong in acute disease but much weaker in chronic infection, might be boosted through and/or synergize with antiviral therapy (19, 21, 40) and therefore mediate viral clearance and prevent relapse at the end of treatment. However, recent studies have been controversial in their findings (28) for both CD4+ (21, 32) and CD8+ (32, 41) T cells and their relationship to treatment outcome of acute HCV infection.
We present here a comprehensive analysis of T-cell responses in acute HCV infection in a group of patients who subsequently received early therapy. In contrast to the earlier studies noted above, we examined immune responses by multiple parameters and at an individual epitope level, using assays based on function as well as direct visualization of T cells.
MATERIALS AND METHODS
Study subjects. Eight subjects with acute HCV infection were recruited in Boston (A1 to A5, A7, and A8) and in Bochum, Germany (A6). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval from the local institutional review boards. HCV seropositivity was defined as the confirmed presence of anti-HCV antibody (third-generation enzyme immunoassay). PCR positivity was defined as the detection of HCV RNA by PCR (detection limit, 600 HCV RNA IU/ml of plasma; version 2.0 Amplicor assay; Roche Diagnostics, Somerville, NJ). Diagnosis of acute HCV infection was based on seroconversion for HCV antibodies and the presence of HCV RNA by PCR. All subjects were negative for HIV antibodies and HBV surface antigen. Additional clinical information about the individuals and the treatments they received is given in Table 1.
PBMC preparation. Fresh peripheral blood mononuclear cells (PBMC) from all study subjects were obtained from centrifugation of blood (drawn into heparin or acid citrate dextrose tubes) over Ficoll solution (Sigma-Aldrich) and washed three times (5 min; 1,500 rpm; 25°C) in RPMI 1640 (Sigma-Aldrich) supplemented with 50 U/ml penicillin, 2 mmol/liter L-glutamine, and 50 μg/ml streptomycin. Cells were processed fresh or frozen and stored in liquid nitrogen.
HCV-derived peptides and recombinant proteins. Peptides corresponding to the amino acid sequence of the HCV-1a strain, spanning the entire HCV polypeptide, were synthesized as free acids using the 9-fluorenylmethoxy carbonyl method. The 301 peptides used in the initial screening assay were 20 amino acids (aa) in length, overlapping adjacent peptides by 10 aa. The 83 optimal epitope peptides were 8 to 10 amino acids in length. Additional truncated peptides were synthesized to determine the optimal epitope sequence.
The recombinant HCV proteins used in this study were expressed as carboxy-terminal fusion proteins with human superoxide dismutase in Saccharomyces cerevisiae or Escherichia coli and were kindly provided by Michael Houghton (Chiron Corporation, Emeryville, Calif.). These proteins were derived from the HCV-1 sequence and encoded core (C22-3 aa 2 to 120), NS3 (C33C aa 1192 to 1457), NS4 (C100 aa 1569 to 1931), NS3/NS4 (C200 aa 1192 to 1931), and NS5 (NS5 aa 2054 to 2995).
HLA typing. HLA typing was performed by the Tissue Typing Laboratory at the Churchill Hospital, Oxford, and the Massachusetts General Hospital Tissue Typing Laboratory using standard molecular techniques (4).
ELIspot assay. Polyvinylidene 96-well plates (Millipore, Billerica, MA) were coated with 2.5 μg/ml recombinant human anti-IFN- antibody (Endogen; Pierce Biotechnology, Rockford, IL) in phosphate-buffered saline (PBS) at 4°C overnight. Fresh or previously frozen PBMC were added at 200,000 cells/well in 140 μl R10 medium (RPMI 1640 [Sigma-Aldrich Corp., St. Louis, MO], 10% fetal calf serum [FCS; Sigma-Aldrich], and 10 mM HEPES buffer [Sigma-Aldrich] with 2 mM glutamine and antibiotics [50 U/ml penicillin-streptomycin]). Peptides were added directly to the wells at a final concentration of 10 μg/ml. The plates were incubated for 18 h at 37°C, 5% CO2. Plates were then washed, labeled with 0.25 μg/ml biotin-labeled anti-IFN- (Endogen), and developed by incubation with streptavidin-alkaline phosphatase (Bio-Rad Laboratories, Hercules, CA) followed by incubation with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Bio-Rad) in Tris buffer (pH 9.5). The reaction was stopped by washing with tap water, and the plates were dried prior to counting on an ELIspot reader (AID, Strassberg, Germany). For quantitation of ex vivo responses, the assay was performed in duplicate, and background was not more than 15 spot-forming cells (SFC)/106 PBMC. Responses were considered positive if the number of spots per well minus the background was at least 25 SFC/106 PBMC (23). Phytohemagglutinin served as a positive control for T-cell stimulation.
HLA class I-peptide tetramer staining. HLA class I-peptide tetramers were prepared as previously described (2) and included tetramers specific for three HCV epitopes restricted by HLA A2 and one HCV epitope restricted by HLA A1, HLA B8, and HLA B35, respectively (for HLA A2, NS3 peptide 1073-1081 [CINGVWCTV], NS4 peptide 1406-1415 [KLVALGINAV], and NS5B peptide 2594-2603 [ALYDVVTKL]; for HLA A1, NS3 peptide 1435-1443 [ATDALMTGY]; for HLA B8, NS3 1395-1367 [HSKKKCDEL]; and for HLA B35, NS3 1359-1367 [HPNIEEVAL]). A total of 0.5 to 1 million PBMC were stained as described (26). Briefly, tetramer staining was performed for 20 min at 37°C. After washing for 5 min with PBS containing 1% FCS at room temperature, cells were pelleted and directly stained with CD8-peridinin chlorophyll protein (Becton Dickinson, Mountain View, CA) for 20 min at 4°C. Cells were then washed as described above and fixed using PBS-1% formaldehyde. Flow cytometric analysis was performed with a BD FACSCalibur, and data analysis was performed using the CellQuest (BD) software. Staining was considered positive if tetramer-positive cells formed a cluster distinct from the tetramer-negative CD8+ T-cell population and the frequency of tetramer-positive cells was greater than 0.02% of the total CD8+ population.
Bulk stimulation of peripheral blood mononuclear cells. In order to establish CD8+ T-cell lines, cryopreserved or fresh PBMC (4 x 106 to 10 x 106) were stimulated with 1 μg/ml of synthetic HCV peptide and 0.5 μg/ml of the anti-CD28 and anti-CD49d (BD) antibodies in R10 (RPMI 1640-10% FCS; Sigma-Aldrich). Irradiated feeder cells (20 x 106 allogeneic PBMC) were added to the culture in a 25-cm2 culture flask (Costar, Cambridge, Mass.). Recombinant interleukin-2 (25 IU/ml) was added on day 2 and twice a week thereafter.
Proliferation assays. Lymphocyte proliferation assays were performed using the HCV proteins described above at concentrations of 10 μg/ml. Fresh PBMC were used for most assays, but selected assays were also performed using frozen cells (as marked in Fig. 4, below). PBMC were plated at 100,000 cells/well in 96-well U-bottom plates (Costar) in 200 μl of R10-HAB medium (RPMI 1640-10% human AB serum) and 10 mM HEPES buffer (Sigma-Aldrich) with 2 mM glutamine and antibiotics (penicillin-streptomycin; 50 U/ml; Sigma-Aldrich) and the designated proteins in quadruplicate wells. After a 6-day incubation at 37°C and 5% CO2, wells were pulsed for 6 h with 1 μCi of [3H]thymidine (NEN, Perkin-Elmer, Boston, MA). Cells were then collected on filters, and the amount of incorporated radiolabel was measured with a beta counter. For the purposes of data interpretation, a stimulation index of 5 or more was considered significant.
Statistical analysis. Statistical analysis was performed using GraphPad Prism (GraphPad, San D
Howard Hughes Medical Institute, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 02129
Nuffield Department of Clinical Medicine, Peter Medawar Building, University of Oxford, United Kingdom
CNRS-BioMerieux, 69365 Lyon, France
Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 02114
Medizinische Universitaetsklinik, Berufsgenossenschaftliche Kliniken Bergmannsheil, 44789 Bochum, Germany
ABSTRACT
Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.
INTRODUCTION
Hepatitis C virus (HCV) infection affects 170 million people worldwide and represents a major public health problem in many countries (24). After acute infection the majority of individuals develop chronic disease, which may result in hepatic failure and liver cancer (34). Persons who clear HCV infection spontaneously usually display vigorous and multispecific cellular immune responses (8, 14, 26, 37), but the mechanisms by which the virus evades immune responses in humans developing chronic infection remain unclear. CD8+ T cells are critical in antiviral defense (35), playing a crucial role in a number of acute and persistent virus infections, such as influenza virus (12), human immunodeficiency virus (HIV) (16, 30, 33), Epstein-Barr virus (EBV) (5, 6, 36), and cytomegalovirus (15). In the chimpanzee model strong and multispecific CD8 T-cell responses have been associated with spontaneous control of HCV (8), and the emergence of escape mutations has been associated with the development of viral persistence (11, 38). Studies addressing this important question in humans are limited, often to responses restricted by a single allele, such as HLA A2 (18, 25, 37, 39), and the critical relationship between the breadth and magnitude of CD8+ T-cell responses in the acute phase of infection and disease outcome remains to be defined.
Once chronic HCV infection is established, therapy with alpha interferon (IFN-) and ribavirin leads to successful virological outcomes in about 50 to 75% of cases (13, 27), depending on viral genotype and pretreatment viral loads. Recent studies have suggested that therapy during the acute phase of infection is much more effective, with up to 95% of patients becoming virus-free after a relatively short course of alpha interferon monotherapy (19, 29). It has been suggested that cellular immune responses, which are strong in acute disease but much weaker in chronic infection, might be boosted through and/or synergize with antiviral therapy (19, 21, 40) and therefore mediate viral clearance and prevent relapse at the end of treatment. However, recent studies have been controversial in their findings (28) for both CD4+ (21, 32) and CD8+ (32, 41) T cells and their relationship to treatment outcome of acute HCV infection.
We present here a comprehensive analysis of T-cell responses in acute HCV infection in a group of patients who subsequently received early therapy. In contrast to the earlier studies noted above, we examined immune responses by multiple parameters and at an individual epitope level, using assays based on function as well as direct visualization of T cells.
MATERIALS AND METHODS
Study subjects. Eight subjects with acute HCV infection were recruited in Boston (A1 to A5, A7, and A8) and in Bochum, Germany (A6). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval from the local institutional review boards. HCV seropositivity was defined as the confirmed presence of anti-HCV antibody (third-generation enzyme immunoassay). PCR positivity was defined as the detection of HCV RNA by PCR (detection limit, 600 HCV RNA IU/ml of plasma; version 2.0 Amplicor assay; Roche Diagnostics, Somerville, NJ). Diagnosis of acute HCV infection was based on seroconversion for HCV antibodies and the presence of HCV RNA by PCR. All subjects were negative for HIV antibodies and HBV surface antigen. Additional clinical information about the individuals and the treatments they received is given in Table 1.
PBMC preparation. Fresh peripheral blood mononuclear cells (PBMC) from all study subjects were obtained from centrifugation of blood (drawn into heparin or acid citrate dextrose tubes) over Ficoll solution (Sigma-Aldrich) and washed three times (5 min; 1,500 rpm; 25°C) in RPMI 1640 (Sigma-Aldrich) supplemented with 50 U/ml penicillin, 2 mmol/liter L-glutamine, and 50 μg/ml streptomycin. Cells were processed fresh or frozen and stored in liquid nitrogen.
HCV-derived peptides and recombinant proteins. Peptides corresponding to the amino acid sequence of the HCV-1a strain, spanning the entire HCV polypeptide, were synthesized as free acids using the 9-fluorenylmethoxy carbonyl method. The 301 peptides used in the initial screening assay were 20 amino acids (aa) in length, overlapping adjacent peptides by 10 aa. The 83 optimal epitope peptides were 8 to 10 amino acids in length. Additional truncated peptides were synthesized to determine the optimal epitope sequence.
The recombinant HCV proteins used in this study were expressed as carboxy-terminal fusion proteins with human superoxide dismutase in Saccharomyces cerevisiae or Escherichia coli and were kindly provided by Michael Houghton (Chiron Corporation, Emeryville, Calif.). These proteins were derived from the HCV-1 sequence and encoded core (C22-3 aa 2 to 120), NS3 (C33C aa 1192 to 1457), NS4 (C100 aa 1569 to 1931), NS3/NS4 (C200 aa 1192 to 1931), and NS5 (NS5 aa 2054 to 2995).
HLA typing. HLA typing was performed by the Tissue Typing Laboratory at the Churchill Hospital, Oxford, and the Massachusetts General Hospital Tissue Typing Laboratory using standard molecular techniques (4).
ELIspot assay. Polyvinylidene 96-well plates (Millipore, Billerica, MA) were coated with 2.5 μg/ml recombinant human anti-IFN- antibody (Endogen; Pierce Biotechnology, Rockford, IL) in phosphate-buffered saline (PBS) at 4°C overnight. Fresh or previously frozen PBMC were added at 200,000 cells/well in 140 μl R10 medium (RPMI 1640 [Sigma-Aldrich Corp., St. Louis, MO], 10% fetal calf serum [FCS; Sigma-Aldrich], and 10 mM HEPES buffer [Sigma-Aldrich] with 2 mM glutamine and antibiotics [50 U/ml penicillin-streptomycin]). Peptides were added directly to the wells at a final concentration of 10 μg/ml. The plates were incubated for 18 h at 37°C, 5% CO2. Plates were then washed, labeled with 0.25 μg/ml biotin-labeled anti-IFN- (Endogen), and developed by incubation with streptavidin-alkaline phosphatase (Bio-Rad Laboratories, Hercules, CA) followed by incubation with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Bio-Rad) in Tris buffer (pH 9.5). The reaction was stopped by washing with tap water, and the plates were dried prior to counting on an ELIspot reader (AID, Strassberg, Germany). For quantitation of ex vivo responses, the assay was performed in duplicate, and background was not more than 15 spot-forming cells (SFC)/106 PBMC. Responses were considered positive if the number of spots per well minus the background was at least 25 SFC/106 PBMC (23). Phytohemagglutinin served as a positive control for T-cell stimulation.
HLA class I-peptide tetramer staining. HLA class I-peptide tetramers were prepared as previously described (2) and included tetramers specific for three HCV epitopes restricted by HLA A2 and one HCV epitope restricted by HLA A1, HLA B8, and HLA B35, respectively (for HLA A2, NS3 peptide 1073-1081 [CINGVWCTV], NS4 peptide 1406-1415 [KLVALGINAV], and NS5B peptide 2594-2603 [ALYDVVTKL]; for HLA A1, NS3 peptide 1435-1443 [ATDALMTGY]; for HLA B8, NS3 1395-1367 [HSKKKCDEL]; and for HLA B35, NS3 1359-1367 [HPNIEEVAL]). A total of 0.5 to 1 million PBMC were stained as described (26). Briefly, tetramer staining was performed for 20 min at 37°C. After washing for 5 min with PBS containing 1% FCS at room temperature, cells were pelleted and directly stained with CD8-peridinin chlorophyll protein (Becton Dickinson, Mountain View, CA) for 20 min at 4°C. Cells were then washed as described above and fixed using PBS-1% formaldehyde. Flow cytometric analysis was performed with a BD FACSCalibur, and data analysis was performed using the CellQuest (BD) software. Staining was considered positive if tetramer-positive cells formed a cluster distinct from the tetramer-negative CD8+ T-cell population and the frequency of tetramer-positive cells was greater than 0.02% of the total CD8+ population.
Bulk stimulation of peripheral blood mononuclear cells. In order to establish CD8+ T-cell lines, cryopreserved or fresh PBMC (4 x 106 to 10 x 106) were stimulated with 1 μg/ml of synthetic HCV peptide and 0.5 μg/ml of the anti-CD28 and anti-CD49d (BD) antibodies in R10 (RPMI 1640-10% FCS; Sigma-Aldrich). Irradiated feeder cells (20 x 106 allogeneic PBMC) were added to the culture in a 25-cm2 culture flask (Costar, Cambridge, Mass.). Recombinant interleukin-2 (25 IU/ml) was added on day 2 and twice a week thereafter.
Proliferation assays. Lymphocyte proliferation assays were performed using the HCV proteins described above at concentrations of 10 μg/ml. Fresh PBMC were used for most assays, but selected assays were also performed using frozen cells (as marked in Fig. 4, below). PBMC were plated at 100,000 cells/well in 96-well U-bottom plates (Costar) in 200 μl of R10-HAB medium (RPMI 1640-10% human AB serum) and 10 mM HEPES buffer (Sigma-Aldrich) with 2 mM glutamine and antibiotics (penicillin-streptomycin; 50 U/ml; Sigma-Aldrich) and the designated proteins in quadruplicate wells. After a 6-day incubation at 37°C and 5% CO2, wells were pulsed for 6 h with 1 μCi of [3H]thymidine (NEN, Perkin-Elmer, Boston, MA). Cells were then collected on filters, and the amount of incorporated radiolabel was measured with a beta counter. For the purposes of data interpretation, a stimulation index of 5 or more was considered significant.
Statistical analysis. Statistical analysis was performed using GraphPad Prism (GraphPad, San D