Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection

Suppression of HCV-Specific T Cells Without Differential Hierarchy Demonstrated Ex Vivo in Persistent HCV Infection Kazushi Sugimoto,1 Fusao Ikeda,1 Jason Stadanlick,1 Frederick A. Nunes,2 Harvey J. Alter,3 and Kyong-Mi Chang1 Hepatitis C virus (HCV) has a high propensity for persistence. To better define the immunologic determinants of HCV clearance and persistence, we examined the circulating HCVspecific T-cell frequency, repertoire, and cytokine phenotype ex vivo in 24 HCV seropositive subjects (12 chronic, 12 recovered), using 361 overlapping peptides in 36 antigenic pools that span the entire HCV core, NS3-NS5. Consistent with T-cell–mediated control of HCV, the overall HCV-specific type-1 T-cell response was significantly greater in average frequency (0.24% vs. 0.04% circulating lymphocytes, P ⴝ .001) and scope (14/36 vs. 4/36 pools, P ⴝ .002) among the recovered than the chronic subjects, and the T-cell response correlated inversely with HCV titer among the chronic subjects (R ⴝ ⴚ0.51, P ⴝ .049). Although highly antigenic regions were identified throughout the HCV genome, there was no apparent difference in the overall HCV-specific T-cell repertoire or type-1/type-2 cytokine profile relative to outcome. Notably, HCV persistence was associated with a reversible CD4-mediated suppression of HCV-specific CD8 T cells and with higher frequency of CD4ⴙCD25ⴙ regulatory T cells (7.3% chronic vs. 2.5% recovered, P ⴝ .002) that could directly suppress HCV-specific type-1 CD8 T cells ex vivo. In conclusion, we found that HCV persistence is associated with a global quantitative and functional suppression of HCV-specific T cells but not differential antigenic hierarchy or cytokine phenotype relative to HCV clearance. The high frequency of CD4ⴙCD25ⴙ regulatory T cells and their suppression of HCV-specific CD8 T cells ex vivo suggests a novel role for regulatory T cells in HCV persistence. (HEPATOLOGY 2003;38:1437-1448.)

H

epatitis C virus (HCV) is a hepatotropic virus with a high rate of persistence associated with chronic hepatitis, cirrhosis, and hepatocellular carcinoma.1 Unfortunately, there is no effective vaccine Abbreviations: HCV, hepatitis C virus; HLA, human leukocyte antigen; HIV, human immunodeficiency virus; IFN-␥, interferon gamma; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; SFU, spot-forming unit; IL-4, interleukin 4; Tregs, regulatory T cells. From the 1Division of Gastroenterology, Department of Medicine, University of Pennsylvania & Philadelphia Veterans Affairs Medical Center, Philadelphia, PA; 2Pennsylvania Hospital, Philadelphia, PA; and 3Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD. Received June 25, 2003; accepted September 20, 2003. Supported by NIH grants AI47519 and AA12849 and the NIH/NIDDK Center of Molecular Studies in Digestive and Liver Diseases P30DK50306 and its Molecular Biology and Cell Culture Core Facilities. This study was also supported in part by the Public Health Service Research Grant M01-RR00040 from the National Institute of Health. K.S. and F.I. contributed equally to this work. Address reprint requests to: Kyong-Mi Chang, M.D., Department of Medicine, GI Division, University of Pennsylvania & Philadelphia VAMC, A212 Medical Research, PVAMC, University and Woodland Avenues, Philadelphia, PA 19104. E-mail: [email protected]; fax: 215-823-4394. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3806-0017$30.00/0 doi:10.1016/j.hep.2003.09.026

for HCV and antiviral therapy remains suboptimal.2-4 Most studies suggest that cellular immune response is a key determinant in the outcome of HCV infection.5-15 Thus, the precise determination of HCV-specific T-cell frequency, cytokine profile, and antigenic hierarchy relative to virologic outcome is relevant to our understanding of HCV immune pathogenesis and vaccine development. However, these immunologic parameters have been difficult to elucidate, in part due to technical limitations resulting in a partial or even biased view of the virus-specific T-cell response. For example, since CD8 T cells are class I human leukocyte antigen (HLA)-restricted, many studies of HCV-specific CD8 T cells used preselected peptides with classic binding motifs for highly prevalent HLA types (e.g., HLA-A2).6,9,11,16 By contrast, T-cell epitopes may lack these classical motifs14,17,18 while certain mutable viruses may even adapt to the more common or conserved HLA alleles, making it problematic to focus on only highly prevalent HLA types.19 Furthermore, prolonged in vitro stimulation used to expand T cells in earlier studies may be biased towards T cells that survive the culture conditions and underestimate the true virus-spe1437

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cific T-cell frequency.20,21 Although the ex vivo methods (e.g., major histocompatibility complex–peptide tetramers or elispot) are more direct and sensitive, most antigenic peptides used in these assays originated from studies with the aforementioned limitations (e.g., HLA-restriction, in vitro expansion).22,23 Finally, antigen-specific T cells may have altered tetramer binding.24 Therefore, the features of “successful” cellular immune response in human HCV infection have not been fully defined. Many of the foregoing concerns are resolved by using overlapping peptides spanning whole viral proteins without specific HLA-restriction elements in direct ex vivo assays.14,17,18,25 We adopted this approach to comprehensively examine the circulating HCV-specific T-cell response relative to outcome, using a peptide library spanning the entire HCV core, NS3-NS5 regions. Our results suggest significant differences in HCV-specific Tcell frequency and scope between HCV clearance and persistence, particularly for the CD4 T-cell subset. Importantly, CD4 T cells may even suppress HCV-specific CD8 T cells during HCV persistence, perhaps through the CD4⫹CD25⫹ regulatory T cells.

Patients and Methods Subjects. The subjects were enrolled from the Philadelphia VA Medical Center and the Hospital of University of Pennsylvania, following written informed consent to participate in the study as approved by the local institutional review board. The “recovered” group included 12 healthy HCV-seropositive subjects (R1-12) negative for HCV RNA by qualitative reverse transcription polymerase chain reaction (Roche COBAS Amplicor; Roche Diagnostics, Indianapolis, IN). HCV serotype was tested in 8 subjects using the Murex HCV serotyping assay (kindly performed by Dr. David Parker, Murex Diagnostics, London, UK) and found to be serotype 1, reflecting previous genotype 1 exposure. The “chronic” group included 12 HCV-seropositive patients (C1-12) chronically infected by HCV genotype 1 (InnoLIPA, Innogenetics, Belgium). Exclusion criteria included human immunodeficiency virus (HIV) coinfection; immunosuppressive or antiviral therapy; chronic liver disease due to hepatitis B virus, autoimmune hepatitis, or primary biliary cirrhosis; and conditions precluding phlebotomy. HCV viremia was quantified by quantitative polymerase chain reaction (Roche Monitor) in plasma with and without 10-fold dilution by normal control plasma because some had high titers beyond the linear range. Class I HLA type was serotyped as previously described.9 Class II HLA type was genotyped through the HLA typing laboratories at the National Institutes of Health and at the University

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of Pennsylvania. As shown in Table 1, the recovered and chronic subjects had similar age, gender, and race distribution with overall well-preserved liver function parameters and no signficant differences in HLA distribution. Predominant HCV risk factors among HCV-seropositive subjects were injection drug or cocaine use (n ⫽ 20) followed by transfusion (n ⫽ 3), and needlestick (n ⫽ 1). As negative control for the HCV-seropositive subjects, 12 healthy HCV-seronegative and RNA-negative subjects (N1-12) without known HCV exposure, history of injection drug use, transfusion, or needlestick exposure were recruited. HCV Peptide Library. A total of 361 peptides (15mers, offset by 6 and overlapping by 9 amino acids) spanning 2,180 amino acid residues in HCV polyprotein were synthesized based on the consensus HCV-H (genotype 1a)26 by Chiron Mimotopes (Clayton, Victoria, Australia; Fig. 1). The peptides spanned 72% of HCV polyprotein, including the entire HCV core and NS3-NS5, with the coordinates based on HCV-H.27 Genotype 1 was selected due to its high prevalence in North America. The peptides were divided into 36 pools (10-11 sequential peptides/ pool, as shown in Fig. 1) for immunologic assays. The 15-mers were immunogenic for both CD4 and CD8 T cells in control experiments. Figure 1B shows efficient CD8 T-cell recognition of 15-mers by the NS3-specific CD8 T-cell line in interferon gamma (IFN-␥) elispot. Figure 1C shows that the type-1 T-cell response to a peptide pool reflects responses to individual peptides within the pool. Recombinant HCV Proteins. Genotype 1a– derived recombinant HCV proteins coding for parts of core, NS3-4 and NS5, and control superoxide dismutase protein were kindly provided by Dr. Michael Houghton (Chiron Corporation, Emeryville, CA).5,12 The proliferative and IFN-␥ responses to these proteins were shown to be CD4 T-cell mediated.12,15 Fluorescent Antibodies. CD4-PE, CD8-PerCP, CD25-FITC, CD45 RO-APC, and isotype antibodies (Becton Dickinson, Franklin Lakes, NJ) were used per the manufacturer’s instruction. Isolation of Peripheral Blood Mononuclear Cells and CD4 Depletion. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described12,15 and used whole and after CD4 depletion in complete media. CD4 depletion was achieved by CD4-dynabeads (Dynal Biotech, Lake Success, NY) per the manufacturer’s instruction, with greater than 90% CD4 depletion confirmed by flow cytometry in all cases. Elispot Assay. HCV-specific type-1 T-cell responses ex vivo using IFN-␥ elispot assay were performed as previously described,15 assessing the total (CD4⫹CD8) T-

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Table 1. Patient Characteristics HLA Class I

HLA Class II

ID

Age

Sex

Race*

ALT (IU/L)

Total Bilirubin (mg/dL)

Albumin (g/dL)

Platelets (ⴛ103/mm3)

A

B

Cw

DRB1

DQB1

R01 R02 R03 R04 R05 R06 R07 R08 R09 R10 R11 R12

45 47 44 52 45 40 48 50 43 55 53 60

M M M M F M M M F F M M

C B B B C B C B C C B B

16 32 25 16 17 39 21 10 24 19 34 23

0.9 0.8 0.6 0.8 0.5 0.9 0.5 0.8 0.4 0.4 0.4 0.9

4.7 4.6 4.0 4.5 4.8 4.6 4.0 4.5 4.1 4.0 4.1 4.5

332 154 261 302 345 154 305 218 308 253 267 244

02, 03 02, 24 02, 29 02, 30 23, 26 24,68 02 02,31 03 03, 32 02, 03 01,33

07, 44 18, 41 35, 44 27, 45 13, 39 45,53 42,27 39,52 44 35 07, 44 07,08

05, 07 07, 17 16 02, 05 06, 12 04,16 02,07 07, 16 07 04 05, 07 03

11,15 04,07 04,13 07,16 01,07 01,08 14,18 08,13 01,15

03,06 03,03 03 02,05 02,05 03,05 03,04 04,06 05,06

Mean C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12

49 47 54 44 53 43 56 52 55 44 45 52 54

M M M M M M M M M M M M

B B C B B B B C B B B B

23 65 44 36 23 104 17 33 175 62 58 98 82

0.7 0.8 1.6 0.7 0.8 0.8 0.6 0.4 1.2 1.9 0.7 0.8 1.2

4.4 4.1 3.8 4.0 4.4 4.4 3.8 4.1 4.5 4.1 4.0 4.1 3.9

262 215 251 233 285 237 305 185 302 180 241 209 192

23, 74 01, 02 03 01, 02 02 03, 34 01,68 11, 24 03,74 02 02,68 68,30

07, 14 14, 27 35, 39 14, 53 53 35 70 15, 27 15,21 22 35,70 44,53

07, 08 02, 08 04, 12 04, 08 04 15, 16 02 01 07 04 03 03,04

11,13 04,08 04,16 07,11 13 03,08 07,13 01 07,15 11,13 04,13 07,13

Mean

50

†66

1.0

†4.1

236

ND 11,15

03,06 ND

02,03 03,06 03,05 02,03 06 04,06 02,06 05 02,06 03,06 03,05 02,06

NOTE. The demographic and clinical information as well as HLA class I/II types are shown for the 12 recovered and 12 chronic subjects. They show similar age, sex, and race distributions without significant differences in HLA type. Both groups showed well-preserved liver function, although with statistically significant differences for ALT activity and albumin (P ⬍ .05). Abbreviations: ALT, alanine aminotransferase; ND, not done. *Race: B (black), C (caucasian). †P ⬍ .05 by Mann-Whitney U test.

cell response in whole PBMCs and CD8 T-cell response in CD4-depleted PBMC fraction. Briefly, whole or CD4depleted PBMCs (0.2 million/well) were plated in 96well elispot plates precoated with anti–IFN-␥ and stimulated with each peptide pool (5 ␮mol/L/peptide) in triplicate, without peptide in 9 replicates (negative control) and with 2 mg/mL phytohemagglutinin (PHA) in triplicates (positive control). The plates were developed after 42 hours and analyzed for spot forming units (SFUs) as previously described,15 excluding assays with high background (average ⬎10 SFU/well in negative control wells) or no PHA response. The frequency of type-1 or IFN-␥⫹ T cells specific for each peptide pool was calculated by subtracting the average SFU in negative control wells from the average SFU in stimulated wells and expressed as HCV-specific IFN-␥⫹ SFUs or type-1 T cells/million PBMCs. The peptidespecific IFN-␥⫹ SFUs were from T cells based on control experiments with and without CD3-depletion by CD3dynabeads (Dynal Biotech, Lake Success, NY) (data not

shown). A positive response required at least 2 of 3 wells with SFUs/well greater than 3 SD above mean negative control response. Because CD4 depletion resulted in relative CD8 enrichment, calculation of HCV-specific type-1 CD8 T-cell frequency in CD4-depleted PBMCs included the CD8 enrichment factor (%CD8 in CD4-depleted PBMCs/ %CD8 in total PBMCs) as determined by flow cytometry. For example, with 2-fold CD8 enrichment following CD4 depletion (e.g., 15%CD8 in whole PBMCs, 30%CD8 in CD4-depleted PBMCs), IFN-␥⫹ SFUs in 200,000 CD4-depleted PBMCs/well was divided by 2 to compensate for the 2-fold CD8 enrichment in each well (compared with 200,000 whole PBMCs/well). Thus, for 50 IFN-␥⫹ SFUs/200,000 CD4-depleted PBMCs/well, the result was calculated as (50 IFN-␥⫹ SFUs/0.2 million PBMCs per well)/2 ⫽ 125 IFN-␥⫹ SFUs/million PBMCs. The results from 36 pools were analyzed individually and summed as a “combined” HCV-specific response.

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Fig. 1. HCV peptide pools. (A) The upper figure shows the strategy for the 361 15-mer peptides (serially offset by 6 and sequentially overlapped by 9 amino acids) based on HCV-H (genotype 1a) spanning 2,180 amino acid residues within the entire core and NS3-NS5 regions and mixed consecutively into 36 pools (10-11 peptides/pool). The table shows the individual pools with their corresponding protein, peptide, and amino acid locations. Because sequences from 2 adjacent proteins were included in pools 14 (NS3 ⫹ NS4), 19 (NS4 ⫹ NS5A), and 27 (NS5A ⫹ NS5B), these pools were grouped with their N-terminal protein and counted only once to estimate the overall T-cell responses to each protein. (B) Efficient CD8 T-cell recognition of 15-mer peptides using a CD8 T-cell line specific for the HLA-A2–restricted epitope NS3 1073 (optimum sequence CINGVCWTV) in the IFN-␥ elispot assay with 1,000 effector T cells and 50,000 antigen-presenting cells (allogeneic HLA-A2 ⫹ Epstein-Barr virus–transformed B cell line) in duplicates at varying concentrations of the optimum peptide (B) as well as three 15-mers (A, C, and D) and one 20-mer (E) containing the optimum peptide. CD8 T-cell recognition was as efficient as the optimum epitope for 15-mers A and C but not for the 15-mers D and 20-mer E containing longer additional sequences beyond the optimum epitope. Since peptides A and D represent 2 consecutive peptides in our overlapping strategy (offset by 6 amino acids), our strategy enables detection of CD8 T-cell response to an optimum epitope through one peptide (e.g., peptide A) even if the 2nd peptide (e.g., peptide D) is not immunogenic. (C) The IFN-␥ response to HCV peptide pools reflect responses to individual peptides using 3 T-cell lines specific for peptide pools 1, 3, and 21. These T-cell lines were expanded in vitro by 2 to 3 weeks of stimulation with each peptide pool (5 ␮mol/L) as previously described9,12 and stimulated with the corresponding peptide pools and the individual peptides within each pool (10,000 effector T cells/well with 50,000 autologous PBMCs as antigen presenting cells in duplicates, 5 ␮mol/L peptide concentration). IFN-␥ responses to the pools were associated with IFN-␥ responses to one or more peptides within each pool, indicating that the responses are peptide specific. Response to individual peptides was not observed in the absence of significant response to the peptide pools (data not shown).

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Type-1 CD4 T-cell responses to recombinant HCV proteins and tetanus toxoid were examined as previously described.15 The type-2 HCV-specific T-cell response was examined using elispot assay with monoclonal anti– interleukin 4 (IL-4) (5 ␮g/mL) as primary antibody and biotinylated anti–IL-4 (2 ␮g/mL) as secondary antibody (Pharmingen, San Diego, CA). Analysis of the CD4ⴙCD25ⴙ T-Cell Function Relative to the HCV-Specific T-Cell Response. The effect of CD4⫹CD25⫹ regulatory T cells (Tregs) on the HCVspecific type-1 T-cell response was examined in IFN-␥ elispot using CD4-depleted PBMCs as effectors, cocultured with several “modulator” cell fractions as follows: (#1) CD4⫹ T cells: ⬎95% pure CD4 T cells negatively selected from PBMCs using CD4 Multisort (Miltenyi Biotec, Auburn, CA); (#2) CD4⫹CD25⫹ T cells: #1 fraction further positively selected for CD25⫹ T cells (%CD4⫹CD25⫹: 45%-79%) using anti-CD25 dynabeads (Dynal Biotech, Lake Success, NY); (#3) CD4⫹CD25- T cells: #1 fraction relatively depleted of CD25⫹ T cells after removing CD25⫹ fraction for #2 (%CD4⫹CD25⫹: 5%-13%); (#4) CD4⫺CD8⫺ fraction: PBMCs sequentially depleted of both subsets by CD4 and CD8 dynabeads (%CD4⫹CD25⫹: ⬍1%). 200,000 CD4⫺ effectors were cocultured with 100,000 modulators (2:1 ratio) in all cases, except in one subject with 160,000 effectors and 80,000 modulators due to low CD4⫹CD25⫹ T-cell yield. The final %CD4⫹CD25⫹ T cells/well was calculated based on their frequencies in effector and modulator fractions measured by flow cytometry: (2 ⫻ %effector ⫹ %modulator)/3. The cell mixtures were stimulated in triplicates with and without immunogenic HCV peptide pools and PHA in IFN-␥ elispot. Flow Cytometry. PBMC fractions were stained for surface and intracellular markers per the manufacturer’s instructions, acquired on FACSCaliber (Becton Dickinson, Franklin Lakes, NJ), and analyzed by Cell Quest. The cutoff for each marker was based on the isotype antibody. Statistical Analysis. The mean values for clinical and immunologic parameters were compared using the nonparametric Mann-Whitney U test. The frequency of positive responses was compared using the ␹2 or Fisher’s exact test based on sample size. Correlations between parameters were tested for statistical significance by Spearman rank correlation.

Results HCV-Specific Type-1 T-Cell Response Relative to Virologic Outcome. The HCV-specific total type-1 Tcell response in PBMCs was examined ex vivo in all recov-

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ered (R1-12), chronic (C1-12), and normal controls (N112), using the 36 HCV peptide pools in IFN-␥ elispot assay. As shown in Fig. 2A and C, the HCV-specific T-cell response was significantly greater in the recovered than in the chronic patients, confirming previous reports and validating our method. For example, the combined HCVspecific T-cell frequency for all 36 pools among the recovered subjects averaged at 2,420 SFUs/106 PBMCs or 0.24% circulating lymphocytes and peaked at 8,148 SFUs/106 PBMCs or 0.81% (R1), contrasting significantly with average frequencies of 0.04% among the chronic patients (P ⫽ .001) or 0.07% among normal controls (P ⫽ .005). The HCV-specific T-cell response among the recovered subjects was also remarkably broad (Fig. 2D), simultaneously targeting 14 of 36 pools on average, compared with only 4 of 36 pools among the chronic patients (P ⫽ .002) or 5 of 36 pools among normal controls (P ⫽ .007). The weak type-1 HCV-specific T-cell response in chronic patients was not due to a type-2 cytokine deviation, based on lack of HCV-specific IL-4 response (Table 2). The weak and focused response among the chronic patients was overall similar to that in the normal controls (354 vs. 704 SFUs/106 PBMCs, P ⫽ .64). Interestingly, two normal controls (N4 and N8) displayed vigorous and broad responses indistinguishable from the recovered subjects and absent among the chronic patients (Fig. 2C and D). Since both subjects had been hospital workers (although without known HCV or needlestick exposure), their T-cell responses may reflect prior exposure without persistent infection or antibody response, as previously described in HCV-seronegative individuals.16,28,29 Excluding these 2 individuals, the average type-1 HCV-specific T-cell response for normal controls was lower at 217 SFUs/106 PBMCs but no different from the chronic patients. Taken together, these results provide an estimate of HCV-specific type-1 T-cell frequency and scope relative to outcome, and they support the role of the HCV-specific type-1 T-cell response in HCV clearance. Contribution of CD8 and CD4 T-Cell Subsets in HCV-Specific T-Cell Response. HCV-specific CD8 Tcell response was assessed concurrently in CD4-depleted PBMC fractions in all subjects (Fig. 2B and C). Unlike the total T-cell response, there was no significant difference between the recovered and chronic groups in HCVspecific CD8 T-cell frequency or breadth (Fig. 2C), suggesting that CD4 T cells may account for most of the immunologic differences observed between the 2 groups. Indeed, a marked 10-fold difference was observed between the recovered and chronic patients in their CD4 T-cell response to 3 recombinant HCV proteins (mean 487 vs. 53 SFUs/106 PBMCs, P ⫽ .001; Fig. 3).

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Fig. 2. The frequency and scope of HCV-specific type-1 total and CD8 T-cell response relative to virologic outcome: Type 1 total T-cell response in whole PBMCs (A) and type 1 CD8 T-cell response in CD4depleted PBMCs (B) specific for 36 HCV peptide pools are expressed as HCV-specific IFN-␥⫹ SFUs/106 PBMCs for 12 recovered (R1-12), 12 chronic (C1-12), and 12 normal control (N1-12) subjects (y axis: HCV-specific type-1 T cells per million PBMCs; x axis: HCV peptide pools 1-36). The combined HCVspecific type-1 T-cell response to all 36 peptide pools are shown in panel C for the overall frequency (in IFN-␥⫹ SFUs/106 PBMCs) and in panel D for overall scope (number of antigenic pools per total 36 pools) in the 12 recovered (R), 12 chronic (C), and 12 normal (N) control subjects. The mean values are written below for each group and indicated by horizontal bars with corresponding P values by the Mann-Whitney U test. The recovered subjects display significantly greater frequency and scope for total but not CD8 T-cell response to HCV, compared with the chronic patients and normal controls.

We then examined the relationship between the combined HCV-specific CD8 and total T-cell response (i.e., with and without CD4 depletion). In the recovered group, CD4 depletion resulted in reduced HCV-specific T-cell response in all subjects with an average CD8:total T-cell ratio of 0.4 (Fig. 4). By contrast, most chronic patients (8 of 12) showed no such reduction upon CD4 depletion. In fact, CD4 depletion led to an overall augmentation in HCV-specific T-cell frequency (mean CD8: total T-cell ratio of 1.4, P ⫽ .006) that was particularly dramatic for subject C10 but also for C1 and C9 (Fig. 4). Accordingly, the overall scope of the response increased with CD4 depletion among the chronic patients, whereas it decreased in the recovered subjects (Fig. 2D). These findings remained significant even if subject C10 with the

greatest augmentation was excluded as an outlier (data not shown), suggesting that multi-specific HCV-specific CD8 T cells are present but functionally suppressed by CD4 T cells in HCV persistence. Immunogenic Regions Targeted by Virus-Specific T Cells in HCV Clearance and Persistence. We then explored the antigenic hierarchy of the HCV-specific Tcell response relative to outcome comparing the 36 regions defined by the peptide pools. As shown in Fig. 5, 13 regions were recognized by total T cells in greater than 50% of the recovered subjects (Fig. 5A). Pool no. 3 in core (92%) and pool no. 34 (75%) in NS5B were the 2 most immunogenic regions, followed by 11 others throughout the polyprotein. Notably, 7 of 13 pools were located within HCV core and NS3 regions, suggesting that these

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Table 2. Lack of HCV-Specific Type-2 T-Cell Response in HCV Clearance and Persistence Subject

IFN-␥ⴙ SFU/106 PBMC

IL-4ⴙ (# ⴙpools)

Subject

IFN-␥ⴙ SFU/106 PBMC

IL-4ⴙ (# ⴙpools)

C1 C2 C3 C4 C5 C7 C8 C9 C10 C11 C12

340 (8) 298 (4) 615 (5) 113 (3) 193 (1) 497 (7) 227 (3) 503 (2) 870 (3) 292 (7) 85 (0)

78 (0) 4 (0) 28 (0) 45 (0) 2 (0) 15 (0) 13 (0) 32 (0) 70 (0) 10 (0) 45 (0)

R1 R2 R3 R4 R5 R6 R7 R8 R9 R11 R12

8,048 (17) 2,193 (9) 942 (24) 2,142 (20) 3,268 (19) 4,025 (28) 723 (6) 3,500 (15) 193 (1) 665 (8) 1,054 (5)

5 (0) 96 (0) 3 (0) 32 (0) 118 (0) 4 (0) 76 (0) 3 (0) 1 (0) 7 (0) 19 (0)

NOTE. The combined HCV-specific T-cell IFN-␥ and IL-4 response to all HCV peptide pools is expressed in SFUs/106 PBMCs with the number of positive pools shown in parentheses. Eleven chronic and 11 recovered subjects (all except C6 and R10) were studied. The dominant cytokine profile was type-1 (IFN-␥⫹) irrespective of outcome. Both IFN-␥ and IL-4 elispot assays included PHA-stimulated positive control wells displaying IFN-␥⫹ or IL-4⫹ spots.

proteins are more immunogenic than others. As expected, chronic patients showed globally weak total T-cell responses peaking only at 33% (Fig. 5B) without differential hierarchy compared with recovered subjects (Fig. 5C and D). Following CD4 depletion, 2 of 13 regions (nos. 3 and 29) maintained similar high immunogenicity among the recovered (Fig. 5A) whereas 2 regions (nos. 9 and 29) became more immunogenic (⬎50% response rate) among the chronic patients (Fig. 5B). Interestingly, the HCV-specific CD8 T-cell response pattern correlated significantly between the 2 groups (R ⫽ 0.69, P ⫽ .0001) with almost superimposable graphs (Fig. 5C and D).

Identified through overlapping peptides spanning most of the HCV genome, rather than preselected epitopes, these results define highly immunogenic regions within HCV core and nonstructural proteins. Further, they show a lack of differential antigenic hierarchy relative to outcome and highlight the global HCV-specific T-cell suppression in persistent HCV infection, perhaps mediated through a CD4 T-cell subset. Suppression of HCV-Specific CD8 T Cells by CD4ⴙCD25ⴙ T Cells. We asked if the antiviral CD8 T-cell suppression in the chronic patients may be mediated by the CD4⫹CD25⫹ regulatory T cells (Tregs), a subset of CD4 T cells associated with T-cell suppression.30-32 To this end, CD4⫹CD25⫹ T cells were directly isolated and examined for their ability to suppress freshly isolated autologous HCV-specific CD8 T cells ex vivo in

Fig. 3. Type-1 CD4 T-cell response to recombinant HCV proteins. HCV-specific Th1 response to recombinant proteins expressing parts of HCV core, NS3-4, and NS5 (in dark gray, light gray, and white, respectively) is displayed in the bar graph, showing a significant difference between the recovered and chronic subjects (combined average of 487 vs. 53 per 106 PBMCs, P ⫽ .001; Mann-Whitney U). The y axis shows HCV-specific IFN-␥⫹ SFUs/106 PBMCs and the x axis shows individual subjects. The IFN-␥⫹ SFUs are derived from CD4 T cells, based on previous control experiments with CD4 depletion.

Fig. 4. Comparison of the combined HCV-specific type-1 T-cell response with and without CD4 depletion. The relationship between the combined HCV-specific CD8 T-cell response in CD4-depleted PBMCs and total T-cell response in undepleted PBMCs is expressed as a ratio (CD8: Total), showing that CD4 depletion results in a decreased HCV-specific CD8 T-cell response to 0.4-fold among the recovered, whereas it results in an average 1.4-fold increase among the chronic patients (mean 0.4 vs. 1.4-fold, P ⫽ .006). The augmentation is particularly notable for C1, C9, and C10.

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Fig. 5. Identification of immunogenic regions in HCV without differential hierarchy relative to virologic outcome. The hierarchy for HCV-specific total and CD8 T-cell responsiveness is shown for 36 regions defined by the peptide pools (A) for the 12 recovered and (B) for the 12 chronic patients in bar graphs (y axis as % responder/pool; x axis for HCV peptide pool number and corresponding proteins). Highly immunogenic regions with % responder greater than 50% are highlighted as dark gray bars and labeled with the pool number above. (C) T-cell responsiveness compared with the 36 peptide pools in chronic and recovered subjects with a positive correlation in the total (R ⫽ 0.15) and CD8 (R ⫽ 0.69) T-cell response. The correlation is highly significant for the CD8 T-cell response (P ⫽ .0001) (y axis: % responder among chronic patients for each pool; x axis: % responder in recovered subjects for each pool). (D) The T-cell response pattern compared with the 36 peptide pools between the recovered (white circles) and chronic (gray circles) patients in total (upper graph) and CD8 T cells (lower graph) (y axis: % responder; x axis: HCV peptide pools 1-36). The patterns are almost superimposable for the CD8 T cells.

the IFN-␥ elispot assay. As shown in Fig. 6A and B for patient C1, there was a marked dose-dependent suppression of HCV-specific and nonspecific CD8 T-cell IFN-␥ production by the CD4⫹CD25⫹ T cells but not CD4⫹CD25⫺ or CD4⫺CD8⫺ PBMC fraction. Similar suppression was confirmed in C9 and C10 as well as in 2 recovered subjects (data not shown), consistent with global T-cell suppression associated with Tregs. Further supporting a role for Tregs in HCV persistence, the circulating CD4⫹CD25⫹ Treg frequency was significantly greater in the chronic than the recovered (7.3% vs. 2.5%, P ⫽ .002) or normal control subjects (3.6%, P ⫽ .017) (Fig. 6C). By contrast, the CD8⫹CD25⫹ T-cell frequency was equally low for all groups (data not shown). Although the positively-selected Tregs could also suppress T-cell response to PHA (Fig. 6A), there was no significant difference between the recovered and chronic patients in their responses to PHA or tetanus toxoid in whole PBMCs (data not shown).15 Inverse Relationship Between HCV-Specific Type-1 T Cell Responses and HCV RNA. While the vigorous T-cell response in the recovered but not chronic patients suggested T-cell–mediated control of HCV, we asked if the weak HCV-specific T-cell response in established

chronic HCV infection may still have antiviral activity. Interestingly, comparing the HCV titer to the combined HCV-specific type-1 T-cell frequency for all 36 pools in each patient, we found a significant inverse correlation between these 2 parameters (R ⫽ ⫺0.51, P ⫽ .049) (Fig. 7). This was also apparent in a subgroup analysis based on HCV titers (P ⫽ .019), suggesting at least partial T-cell– mediated control of HCV during persistent infection. HCV-specific T-cell responses did not correlate significantly with clinical liver function or liver histology (data not shown), although the sample size may be too small for such clinical comparisons.

Discussion Mechanisms of viral persistence are multifactorial and involve both viral and host factors. For HCV, persistent infection may be associated with its mutable quasispecies nature33 and immune interference.34-36 Nonetheless, HCV is cleared spontaneously in some persons, particularly in those with a vigorous HCV-specific T-cell response. Thus, cell-mediated immunity is believed to play a critical role in the outcome of HCV infection. In this study, we applied the overlapping peptide approach in

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Fig. 6. Suppression of HCV-specific CD8 T cells by CD4⫹CD25 ⫹ Tregs. (A) The suppression of HCV-specific CD8 T-cell IFN-␥ response by CD4⫹CD25⫹ T cells in IFN-␥ elispot assay for patient C1. In this assay, CD4-depleted CD8-enriched effector cells were cocultured with 3 different modulator cell fractions (CD4⫺CD8⫺, CD4⫹CD25⫺, CD4⫹CD25⫹ fractions as described in the Patients and Methods section) and stimulated with HCV peptide pools ex vivo in triplicates. The %CD4⫹CD25⫹ T cells in parentheses below (0.4%, 3.8%, and 19.1%) reflect the final CD4⫹CD25⫹ T-cell content in each well after the modulator cells were mixed with the effectors at 1:2 ratio. (B) The dose-dependent suppression of HCV-specific CD8 T-cell IFN-␥ response by CD4⫹CD25⫹ T cells in patient C1 with CD4⫹CD25⫹ T-cell frequencies of 0.4% (white bars), 3.8% (gray bars), and 19.1% (black bars) (y axis: number of peptide-specific IFN-␥⫹ T cells/well; x axis: 4 immunogenic HCV peptide pools). (C) The circulating CD4⫹CD25⫹ T-cell frequency relative to outcome in recovered (2.5%), chronic (7.3%), and normal control (3.6%) subjects. The CD4⫹CD25⫹ T-cell frequency is significantly greater among the chronic patients compared with recovered (P ⫽ .002) or normal control subjects (P ⫽ .017) by the Mann-Whitney U test, suggesting an association beween CD4⫹CD25⫹ T cells and HCV persistence.

direct ex vivo cytokine assay to overcome some of the previous limitations and to examine the HCV-specific T-cell response relative to outcome in a more comprehensive fashion. The average circulating HCV-specific type-1 T-cell frequency was 0.24% among the recovered subjects. Perhaps higher than previously estimated, this range of T-cell frequency was not unexpected given our use of the sensitive ex vivo method with peptides spanning most of the HCV polyprotein. The HCV-specific T-cell response among HCV-recovered subjects was also remarkable for its breadth, simultaneously targeting up to 28 of 36 (78%) regions of HCV. By contrast, HCV persistence was associated with a significantly weaker HCV-specific T-cell response with almost 10-fold less frequency and 4-fold less scope compared with the recovered subjects, suggesting that both quantity and scope of the HCVspecific T-cell response determine the outcome of human

Fig. 7. Inverse relationship between the HCV-specific type 1 T-cell response and HCV RNA titer. (A) A significant negative correlation (R ⫽ ⫺0.51, P ⫽ .049 by Spearman rank correlation) between the HCVspecific T-cell response and HCV RNA titer. (B) This relationship in a subgroup analysis based on a cutoff of 2 million IU/mL HCV RNA with significantly greater combined HCV-specific total type 1 T-cell response for subjects with HCV titer below 2 million IU/mL than those with titers greater than 2 million IU/mL (474 vs. 185 HCV-specific IFN-␥ SFUs/106 PBMCs, P ⫽ .019 by the Mann-Whitney U test) (y axis: HCV-specific type 1 total T-cell frequency in 106 PBMCs; x-axis: HCV RNA titer [⫻ million IU/mL serum].

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HCV infection similar to some but not all chimpanzee studies.10,37-39 Importantly, HCV persistence was not associated with a type-2 switch in cytokine phenotype. The weak circulating HCV-specific T-cell response in chronic patients was nonetheless inversely associated with viral titer, suggesting that these peripheral T cells are virologically relevant. Our results are also consistent with previous studies (albeit with limited antigenic repertoire and in vitro expanded T-cell lines) and the high HCV titers observed in immunosuppressed patients.6,40 Inverse relationships between the virus and virus-specific T cells have also been reported in HIV,41,42 although a recent study using a similar comprehensive overlapping peptide approach showed a remarkably high HIV-specific type-1 T-cell frequency (median 10,640 SFUs/106 PBMCs) without correlation with HIV titer in chronic untreated HIV-1 infection.25 Based on this contrast, one could speculate that HCV is less immunogenic than HIV, yet more responsive to virus-specific T cells and type-1 cytokines, striking a hopeful note for T-cell– based therapeutic strategies for HCV. As for the antigenic hierarchy, both HCV core and NS3 regions were highly immunogenic among the recovered but not chronic patients. On the whole, 13 pools were immunogenic in greater than 50% of recovered subjects, defining 8 discrete, highly immunogenic regions that may be useful in vaccine design, in Core (pools 2-3), NS3 (pools 4, 7, and 10-12), NS4 (pools 16-17), NS5A (pools 19-20), and NS5B (pools 29 and 34). Interestingly, the similar antigenic repertoire between the recovered and chronic subjects (particularly the CD8 T-cell response) suggested that HCV persistence is not due to a differential antigenic hierarchy but rather a global HCVspecific T-cell suppression. One notable finding in this study was the differential contribution of CD4 and CD8 T-cell subsets to the HCV-specific T-cell response relative to virologic outcome. Among recovered subjects, CD4 T-cell depletion resulted in reduced HCV-specific T-cell response by about half, suggesting contribution by both T-cell subsets. However, in the chronic patients, CD4 T cells contributed minimally to the total HCV-specific T-cell response while their depletion unmasked significant HCV-specific CD8 T-cell response with frequency, scope, and repertoire similar to the recovered subjects. This was not due to technical variations in CD4 T-cell depletion or single outlier (e.g., C10). The similarity in the type-1 CD8 T-cell response between the patient groups is consistent with our previous report using intracellular IFN-␥ staining ex vivo (with a limited number of known CD8 T-cell epitopes),12 although we did not address the cytolytic capacity of in vitro expanded HCV-

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specific CD8 T cells in the current study. Taken together, these results suggest that HCV-specific CD8 T cells are maintained but functionally suppressed during chronic HCV infection perhaps through a CD4 T-cell–mediated mechanism. We considered the immunoregulatory CD4⫹CD25⫹ T cells (Tregs) as a potential candidate for this suppressive CD4 T-cell subset. Recently under intense reinvestigation, these regulatory T cells expressed IL-2 receptor ␣-chain (CD25) and mediated cell-cell contact-dependent T-cell suppression through various potential mechanisms including IL-10, transforming growth factor ␤, and IL-6, contributing to self-tolerance, autoimmunity, and pathogen- or tumor-specific immune response.30,31,43 Indeed, Tregs from chronic patients could directly suppress HCV-specific CD8 T-cell IFN-␥ production ex vivo, consistent with our hypothesis. Although Tregs from recovered persons also suppressed the HCV-specific CD8 T-cell IFN-␥ response, CD4 depletion did not augment the HCV-specific T-cell response in any of them. Importantly, Treg frequencies were significantly higher among the chronic patients compared with recovered or normal control subjects. Although CD25 is also a marker of activated T cells, increased CD25 expression was limited to the CD4 but not CD8 T cells, suggesting that this is not nonspecific activation due to ongoing hepatitis. While we could not directly examine the effect of Tregs on HCV-specific CD4 T cells, it is likely that Tregs also suppress HCV-specific CD4 T cells and contribute to further immune dysregulation. Interestingly, the increased Treg frequency was not associated with clinically apparent immune defects or T-cell responses to PHA or tetanus toxoid in the chronic patients, suggesting that HCV-specific T cells may be particularly susceptible to Treg-mediated suppression. Our findings are notable in the context of virus-specific T-cell dysfunction reported in HCV persistence,15,22,44 including the study by Ulsenheimer et al. in which CD25 expression after brief in vitro HCV antigen stimulation was used to identify HCV-specific CD4 T cells.45 Although the CD25 expression in that study was induced by antigenic stimulation in vitro (unlike the “naturally occurring” Tregs in our study in which CD25 expression was detected directly without in vitro stimulation), it is conceivable that the Tregs may be HCV-specific or induced by HCV variants or T-cell antagonists, particularly given the increased Treg frequency in HCV persistence. HCV may also promote dendritic cell dysfunction,46,47 natural killer cell inhibition, T-cell activation,34,35 transcriptional activation of IL-2 promoter,48 and Tr1 cells with regulatory cytokine IL-10 production.49 Furthermore, Tregs can be induced by repetitive allostimulation with imma-

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ture but not mature dendritic cells in vitro.50 Thus, it is tempting to speculate that HCV may promote its own survival by up-regulating Tregs that suppress the HCVspecific T cells. Along these lines, although the mechanisms of Treg-mediated T-cell suppression are not clarified, blocking the Treg-mediated suppression of HCV-specific T cells (either alone or with vaccine targeting highly immunogenic regions) may provide therapeutic virus control. In conclusion, we showed that the vigor and scope of HCV-specific type-1 T-cell response (but not antigenic repertoire) are important determinants of HCV clearance. By contrast, HCV persists in the setting of globally suppressed antiviral T-cell response associated with high frequency of CD4⫹CD25⫹ regulatory T cells that can suppress HCV-specific T cells ex vivo. These results define some of the immunologic features relevant in HCV infection and suggest a novel mechanism of HCV persistence that warrants further investigation. Acknowledgment: The authors thank Drs. Carl June, Hao Shen, Francis V. Chisari, and Thomas Judge for thoughtful discussions and careful reading of this manuscript as well as Dr. Michael Houghton at Chiron Corporation for generous provision of the recombinant HCV antigens. They gratefully acknowledge Mary Valiga, RN, for patient recruitment and sample processing as well as the study subjects who participated in this study both at the Philadelphia Veterans Affairs Medical Center clinics, Hospital of University of Pennsylvania, and the NIHfunded Clinical Research Center within the University of Pennsylvannia. They also acknowledge the kind support for HLA genotyping by Sharon Adams, MT, ASCP, at the HLA laboratory in the Department of Transfusion Medicine at the National Institutes of Health and Dr. Malek Kamoun at the HLA laboratory at the University of Pennsylvania.

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