Regulatory T Cells Suppress In Vitro Proliferation of Virus-Specific CD8+ T Cells during Persistent Hepatitis C Virus Infection

The basis of chronic infection following exposure to hepatitisC virus (HCV) infection is unexplained. One factor may be thelow frequency and immature phenotype of virus-specific CD8⁺T cells. The role of CD4⁺CD25⁺ T regulatory (T_reg) cells inpriming and expanding virus-specific CD8⁺ T cells was investigated.Twenty HLA-A2-positive patients with persistent HCV infectionand 46 healthy controls were studied. Virus-specific CD8⁺ T-cellproliferation and gamma interferon (IFN- ${gamma}$ ) frequency were analyzedwith/without depletion of T_reg cells, using peptides derivedfrom HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV).CD4⁺CD25⁺ T_reg cells inhibited anti-CD3/CD28 CD8⁺ T-cell proliferationand perforin expression. Depletion of CD4⁺CD25⁺ T_reg cells fromchronic HCV patients in vitro increased HCV and EBV peptide-drivenexpansion (P = 0.0005 and P = 0.002, respectively) and alsothe number of HCV- and EBV-specific IFN- ${gamma}$ -expressing CD8⁺ T cells.Although stimulated CD8⁺ T cells expressed receptors for transforminggrowth factor beta and interleukin-10, the presence of antibodyto transforming growth factor beta and interleukin-10 had noeffect on the suppressive effect of CD4⁺CD25⁺ regulatory T cellson CD8⁺ T-cell proliferation. In conclusion, marked CD4⁺CD25⁺regulatory T-cell activity is present in patients with chronicHCV infection, which may contribute to weak HCV-specific CD8⁺T-cell responses and viral persistence.

INTRODUCTION

Top

Introduction

There are 170 million patients worldwide with persistent hepatitisC virus (HCV) infection who are at significant risk of progressiveliver injury leading to cirrhosis, death from liver failure,and liver cancer (2, 11, 22). There is a crucial need to delineatethe underlying pathogenesis. One important aspect of persistentHCV infection may be the low frequency of circulating HCV-specificCD8⁺ T cells (13, 23), despite high-level viral replicationand viral protein production. This is in marked contrast toacute infection, where the CD8⁺ T-cell response may comprise8% of total CD8⁺ T cells and include responses to at least eightseparate epitopes (24). If viremia persists after acute infection,CD8⁺ T-cell responses wane rapidly (23).

It has been argued that T-cell exhaustion (20) or viral mutation(12, 20) may cause the low number of circulating antigen-specificCD8⁺ T cells during HCV infection. The failure of HCV-specificCD8⁺ T cells to expand may be important in viral persistence,as it has been recently shown that a strong CD8⁺ T-cell responsein the chimpanzee model correlates with protection against reinfectionand is associated with viral clearance during primary infection(37). Furthermore, in nonviremic HCV antibody-positive patients,HCV-specific CD8⁺ T cells expand more readily compared to viremicpatients (39).

Few studies have addressed the reasons for either the low frequencyof HCV-specific CD8⁺ T cells or the immature phenotype associatedwith persistent HCV viremia (27). One possible mechanism maybe regulation by CD4⁺CD25⁺ regulatory T cells (T_reg) (38). Arecent study has shown an increase in CD4⁺CD25⁺ T_reg cells duringchronic HCV infection and that these cells may secrete interleukin(IL)-10 and transforming growth factor beta (TGF-ß)(7). In addition, HCV-specific gamma interferon (IFN- ${gamma}$ ) secretionby peripheral blood mononuclear cells (PBMC) could be recoveredor increased by the addition of anti-TGF-ß.

Secondary memory CD8⁺ T-cell responses in a murine model arecontrolled by CD4⁺CD25⁺ T_reg cells (30). This is in contrastto primary immune responses, which are dependent on CD4⁺ T-cellhelp (19). In murine and human studies, CD4⁺CD25⁺ T_reg cellshave been characterized by intracellular expression of cytotoxicT-lymphocyte-associated antigen 4 (CTLA-4) and have importantroles in the prevention of autoimmune disease (29, 35, 36) andtransplantation tolerance (9, 17, 33, 41). Secreted IL-10 andmembrane-bound TGF-ß may both be critical to CD4⁺CD25⁺T_reg cell function (14, 15), although in vitro data are inconclusive(8). We addressed the hypothesis that HCV-specific CD8⁺ T-cellexpansion in chronic HCV infection may be controlled by CD4⁺CD25⁺T_reg cells.

MATERIALS AND METHODS

Top

Materials and Methods

Study subjects and samples. Twenty HCV antibody-positive, HCV RNA-positive, HLA-A2-positivepatients were selected from a cohort of outpatients in Cambridge,United Kingdom. Peripheral blood mononuclear cells (PBMC) wereobtained from patients by centrifugation of heparinized bloodover Lymphoprep (Nycomed, Roskilde, Denmark). PBMC were alsoisolated from six HLA-A2-positive healthy individuals with serologicalevidence of past cytomegalovirus (CMV) and/or Epstein-Barr virus(EBV) infection. All samples were obtained with approval ofthe Local Ethics Research Committee. Patient details are shownin Table 1.

View this table:
[in this window]
[in a new window]
TABLE 1. Chronically HCV-infected patient data

Cell isolation. In the initial regulatory T-cell characterization experimentsin Fig. 1 and 2, CD4⁺CD25⁺ cells were first isolated by negativedepletion of CD4⁺ T cells from PBMC and the CD25⁺ T cells werepurified from this CD4⁺ fraction by magnetic depletion usingmouse anti-human CD25⁺ beads as per the manufacturer's instructions(Miltenyi Biotec, Bergisch Gladbach, Germany) and separatedinto CD25⁺ (referred to as CD4⁺CD25⁺) or CD25^– cells (referredto as CD4⁺CD25^–). All further experiments were performedusing CD25⁺ T cells isolated from PBMC (also referred to asCD4⁺CD25⁺) by magnetic depletion using mouse anti-human CD25⁺beads. Fluorescence-activated cell sorting analysis after depletionshowed that over 85% of the isolated CD25⁺ cells were CD4⁺.In the CD25⁺-depleted PBMC fraction, <5% of the originalCD25⁺ population was detectable. In addition, CD8⁺ T cells wereisolated using mouse anti-human CD8⁺ beads (Miltenyi Biotec).

View larger version (42K):
[in this window]
[in a new window]
FIG. 1. Function and phenotype of CD4⁺CD25⁺ cells from patients with persistent HCV infection. (A) CD4⁺CD25⁺ T_reg cells, CD4⁺CD25^– T cells, or a combination of these at a ratio of 1:1 were stimulated with anti-CD3 and cell proliferation was measured using [³H]thymidine incorporation. (B and C) Intracellular CTLA-4 expression of (B) CD4⁺CD25⁺ cells and (C) CD4⁺CD25^– cells. (D) Percentage of CD8⁺ and CD4⁺CD25⁺ cells within the intrahepatic lymphocyte population. (E) Histological staining for Foxp3 within the liver of an HCV-infected individual.

View larger version (18K):
[in this window]
[in a new window]
FIG. 2. Inhibition of CD8⁺ T-cell proliferation and perforin expression by CD4⁺CD25⁺ T_reg cells. (A) CD8⁺ T cells or (B) CD8⁺ T cells in the presence of CD4⁺CD25⁺ T_reg cells were stimulated with anti-CD3/CD28 and stained with anti-Ki-67, a nuclear marker of proliferation. Histograms are gated on CD8⁺ T cells. (C) The proportion of Ki-67-positive CD8⁺ T-cell was reduced in the presence of CD4⁺CD25⁺ T_reg cells (coculture; n = 6) and was not restored upon addition of anti-IL-10 or anti-TGF-ß antibodies. (D) Intracellular perforin expression of anti-CD3/CD28 activated CD8⁺ T cells with (grey filled) or without CD4⁺CD25⁺ T_reg cells (black line) at a 1:1 ratio. (E) The proportion of intracellular perforin-positive CD8⁺ T cells was reduced in the presence of CD4⁺CD25⁺ T_reg cells (coculture; n = 6). Horizontal bars represent median values, the box represents the interquartile range, and the error bars represent the range.

Flow cytometric analysis. The following conjugated antibodies and additional fluorometricreagents were used: CD4-fluorescein isothiocyanate (FITC)/CyChrome,CD8-CyChrome, CD25-phycoerythrin (PE), CTLA-4-CyChrome, Ki-67-FITC,IL-10 receptor-PE (all from BD Pharmingen, Oxford, United Kingdom),TGF-ß receptor type II-FITC, active TGF-ß-biotinylated(R&D Systems, Minneapolis, MN), and streptavidin-PE (BDPharmingen). HLA class I peptide tetramers were prepared aspreviously described (3) and included tetramers specific forfive epitopes restricted by HLA-A2: HCV-NS3 peptides 1073 to1081, CINGVCWTV; HCV-NS4 peptides 1406 to 1415, KLVALGINAV;HCV-NS5b peptides 2594 to 2603, ALYDVVTKL; CMV-pp65 peptides495 to 503 NLVPMVATV; and EBV-BMLF1 peptides 280 to 288, GLCTLVAML.All peptides were synthesized by Research Genetics (Invitrogen,Paisley, United Kingdom). Tetramer staining was performed aspreviously described (26). Cells were acquired on a FACSCaliburand analyzed using Cellquest software (BD Pharmingen). Intracellularstaining was performed according to the manufacturers' instructions(Cytofix/Cytoperm; BD Pharmingen).

Proliferation assays. We plated 10⁵ cells per well of CD4⁺CD25⁺ T_reg, CD4⁺CD25^–and CD8⁺ T cells separately or in specific combinations in a96-well round-bottom plate. Samples were prepared in triplicate,and anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) (BDPharmingen) were added to each well to a total volume of 200µl. Proliferation was then determined on day 5 eitherby thymidine incorporation or by flow cytometric analysis usingKi-67 as a nuclear marker of cell proliferation. Proliferationwas assessed by incorporation of 0.5 µCi/well [³H]thymidine(Amersham Pharmacia, Little Chalfont, United Kingdom) afterculture and 6 h prior to harvesting. PBMC were collected ontofilters using an automatic cell harvester (Wallac, Turku, Finland)and incorporated [³H]thymidine was measured using a ß-counter(Wallac). The results were expressed as counts per minute (cpm)minus the mean cpm for the control wells. Cell counts did notdiffer by more than 10% (n = 6) for the CD4⁺CD25⁺ cells whenperformed on days 0 and 5 after anti-CD3/CD28 treatment.

Peptide restimulation. PBMC, depleted or not depleted of CD25⁺ cells using CD25⁺ beads(Miltenyi Biotec), were restimulated in 48-well plates (5 x10⁵ cells per well) with 10 µg/ml peptide in 1 ml of RPMI1640, 10% human AB serum (First Link, United Kingdom), 100 µg/mlstreptomycin, 100 U/ml penicillin, and 2 mM glutamine (RPMI-10)and incubated at 37°C in a humidified CO₂ (5%) incubator.On days 3 to 4, 500 µl of RPMI-10 containing 200 U/mlof recombinant interleukin-2 (Chiron, Emeryville, CA) was added,and further medium changes with 100 U/ml of recombinant interleukin-2were performed every 3 to 4 days until they were analyzed onday 14. In parallel, CD4⁺CD25⁺ T_reg cell-depleted PBMC wereincubated with the CD4⁺CD25⁺ T_reg cells (termed repletion) atratios of 1:2 and 1:10 (CD4⁺CD25⁺ T_reg cells:depleted PBMC).CD4⁺CD25⁺ T_reg cell repletion was also performed in the presenceof 200 U/ml and 400 U/ml of IL-2 on day 0. After 14 days, thepercentage of tetramer-specific CD8⁺ T cells was determined.Cytokine blocking experiments were also performed using antibodiesto TGF-ß or IL-10 (10 µg/ml) (R&D Systems).

Enzyme-linked immunospot assays. CD4⁺ T-cell IFN- ${gamma}$ ELISpot assays were performed on 2 x 10⁵ PBMCwith or without CD25⁺ cells (n = 6). Cells were preincubatedfor 24 h in the presence of a pool of three HCV peptides, anEBV peptide (10 µg/ml), or phytohemagglutinin (at 1 µg/ml;Sigma, Dorset, United Kingdom). In parallel, 96-well Immobilon-Pmembrane microtiter plates (Millipore, Bedford, MA) were coatedwith 100 µl of 5 µg/ml anti-IFN- ${gamma}$ monoclonal antibodyin a human IFN- ${gamma}$ enzyme-linked immunospot (ELISpot kit; BD Biosciences).These plates were incubated for 16 h at 4°C, after whichthey were washed and then blocked with RPMI-10 for 2 h. Themedium was removed and the preincubated cell cultures were transferredto the Immobilon-P plates and incubated for a further 24 h.Following this, plates were washed with water and then withphosphate-buffered saline (PBS)/0.5% Tween-20; 100 µlof 2 µg/ml detection biotinylated anti-IFN- ${gamma}$ was addedto each well and incubated for 2 h at room temperature. Theplates were washed again, and 100 µl of avidin-horseradishperoxidase conjugate was added to each well and incubated for1 h at room temperature. Unbound conjugate was removed by thoroughwashing, and finally 100 µl of AEC (3-amino-9-ethylcarbazole)substrate was added to each well for exactly 15 min. The reactionwas stopped with extensive washing with water. The plates weredried overnight at room temperature and the spots were countedby the AID ELISpot reader system (AID Diagnostika GmbH, Strassberg,Germany). The number of specific spot-forming cells was determinedas the mean number of spots in the presence of an antigen minusthe mean number of spots in the wells with medium only and expressedper 10⁶ PBMC.

Isolation of intrahepatic lymphocytes. Fresh liver tissue was obtained from six viremic patients atdiagnostic biopsy. Samples were placed in RPMI supplementedwith 10% fetal bovine serum and 0.02% EDTA, disaggregated using21-gauge needles, and the cell suspension was filtered throughnylon mesh. Hepatocytes were removed by centrifugation at 50x g. The supernatant was centrifuged at 2,000 x g and the pelletwas washed twice in PBS and suspended in RPMI. The yield ofmononuclear cells was 1.5 to 5 x 10⁵.

Immunohistochemistry of intrahepatic lymphocytes. Formalin-fixed, paraffin-embedded sections (5 µm) on 3-aminopropyltriethoxysilane(APES)-coated slides were dewaxed in xylene and taken througha series of ethanol washes. The tissues were pressure-cookedin 0.08 M citrate buffer (pH 6.0) for 3 min to facilitate antigenretrieval. Following washes in Tris-buffered saline (TBS), endogenousperoxidase activity was quenched by incubation in 0.6% hydrogenperoxide in TBS and blocked with appropriate serum (10% rabbit)in TBS for 2 h (DAKO, United Kingdom). A goat polyclonal Foxp3(ABCAM, United Kingdom) was applied (100 µl) in a humidifiedchamber at 4°C overnight with gentle shaking in 1% bovineserum albumin/TBS with 0.1% Triton X-100. The slides were washedin TBS containing 0.025% Triton X-100 and incubated for 1 hwith rabbit anti-goat Foxp3. A streptavidin-horseradish peroxidasesystem (DAKO, United Kingdom) with the substrate diaminobenzidinewas used for development. Slides were counterstained with Harrishematoxylin, dehydrated in ethanol, and cleared in xylene. Coverslipswere applied with DPX mounting medium (Gurr, United Kingdom).Omission of the primary antibody served as a negative control.

Statistical analysis. In paired experiments, the distribution of the data was assumedto be nonparametric. The medians of the data sets were thencompared using a Wilcoxon signed rank test. Statistical analysiswas performed on SPSF version 11.5. Significant values (P <0.05) are indicated.

RESULTS

Top

Results

CD4⁺CD25⁺ T_reg cells from patients with HCV do not proliferate and regulate both CD4⁺CD25^– and CD8⁺ T-cell proliferation. To determine whether the CD4⁺CD25⁺ T-cell population had suppressorfunction in persistent HCV infection and whether the cells isolatedin these experiments were comparable with those in other studies,classical T-cell suppression assays were performed in six patients.Isolated CD4⁺CD25⁺ T_reg cells did not proliferate after nonspecificsoluble anti-CD3 stimulation. In contrast, CD4⁺CD25^– Tcells proliferated, and this was abrogated in coculture withCD4⁺CD25⁺ T_reg cells at a 1:1 ratio. (Fig. 1A). In addition,increased expression of intracellular CTLA-4 was present inCD4⁺CD25⁺ T_reg cells (Fig. 1B) compared to CD4⁺CD25^– Tcells (Fig. 1C).

To examine the frequencies of CD25⁺ T cells in liver (a likelysite of activity), the percentage of CD4⁺CD25⁺ T_reg cells (median8.95%) and CD8⁺ T cells (median 17.9%) was determined from liverbiopsies of 6 chronic HCV patients (Fig. 1D). Therefore an approximateratio within the liver of CD4⁺CD25⁺ T_reg cell: CD8⁺ T cellsis 1:2. Foxp3 stained liver histology samples were also analyzed(Fig. 1E), which indicated that CD4⁺CD25⁺ T_reg cells appearto predominate around the portal tract. Therefore, the CD4⁺CD25⁺T_reg cell:CD8⁺ T-cell ratio may vary significantly within variouscompartments of the liver.

To determine whether the CD4⁺CD25⁺ T_reg cells could also suppressthe proliferation of CD8⁺ T cells during persistent HCV infection,the latter were stimulated with anti-CD3/anti-CD28. Proliferationof CD8⁺ T cells, measured by nuclear Ki-67 expression, was inhibitedby coculture with CD4⁺CD25⁺ T_reg cells in a 1:1 ratio (P = <0.05,n = 6; Fig. 2A-B). CD4⁺CD25⁺ T_reg cells also suppressed theexpression of intracellular perforin in activated CD8⁺ T cells(P = <0.05; n = 6; Fig. 2D). Group data of Ki-67 and perforinexpression are shown in Fig. 2C and E, respectively. The inhibitoryeffect of CD4⁺CD25⁺ T_reg cells on CD8⁺ T-cell expansion couldnot be reversed by culture with antibody to either TGF-ßor anti-IL-10 (Fig. 2C).

CD4⁺CD25⁺ T_reg cells regulate proliferation of HCV-specific CD8⁺ T cells. The frequency of HCV-specific CD8⁺ T cells after in vitro expansionwith peptide increased with depletion of CD4⁺CD25⁺ T_reg cells(P = 0.0005, Fig. 3A, top panel; Fig. 3B, top panel summarizesthe 12 individual responses). In 12/20 patients studied, therewas a significant increase (as determined by the size of thetetramer response) in antigen-specific CD8⁺ T-cell expansionto at least one of the three HCV peptides studied followingdepletion of CD4⁺CD25⁺ T_reg cells. In 5 of those 12 patients,the HCV-specific CD8⁺ T-cell response was undetectable afterrestimulation of PBMC until depletion of CD4⁺CD25⁺ T_reg cells.For the 12 individual peptide responses there was a median 43-foldincrease in the size of the tetramer response following depletionof CD4⁺CD25⁺ T_reg cells (P = 0.0005).

View larger version (49K):
[in this window]
[in a new window]
FIG. 3. CD4⁺CD25⁺ T_reg cells suppress the expansion of virus-specific CD8⁺ T cells during HCV infection. Proliferation in response to peptide stimulation was measured by tetramer frequency. Examples of individual patients are shown: (A) PBMC alone (left-hand panels) and CD4⁺CD25⁺ T_reg-depleted PBMC (right-hand panels) were restimulated with HCV-specific (top panels), CMV-specific (middle panels), or EBV-specific (bottom panels) peptides, and the extent of proliferation was measured by tetramer frequency. (B) Overall results are shown for HCV (top panel), CMV (middle panel), and EBV (bottom panel). The left-hand box represents the responses in unfractionated PBMC, the right-hand box represents the responses following depletion of CD4⁺CD25⁺ T_reg cells. Horizontal bars represent median values, the box represents the interquartile range, and the error bars represent the range.

To determine if this effect was HCV specific or extended toother virus-specific CD8⁺ T- cell responses, the effect of CD4⁺CD25⁺T_reg cell depletion on CMV- and EBV-specific CD8⁺ T-cell responseswas studied. Five of the patients had serological evidence ofprevious CMV infection and 15 had serological evidence of previousEBV infection.

The tetramer frequency of CMV-specific CD8⁺ T cells also increased(median, sixfold) in all five CMV-positive individuals followingdepletion of CD4⁺CD25⁺ T_reg cells (Fig. 3A, middle panel; Fig.3B, middle panel summarizes group data) (P = 0.043). The frequencyof EBV-specific CD8⁺ T cells also increased (median 3 fold)in 14/15 patients following depletion of CD4⁺CD25⁺ T_reg cells(Fig. 3A, bottom panel; Fig. 3B, bottom panel summarizes groupdata) (P = 0.002).

Addition of CD4⁺CD25⁺ T_reg cells suppresses antigen-specific CD8⁺ T-cell proliferation. Isolated CD4⁺CD25⁺ T_reg cells were added back to the depletedPBMC population prior to restimulation. Almost total abolitionof HCV-specific CD8⁺ T-cell responses was seen in 11/12 patientsat ratios of 1:2 and 1:10 (CD4⁺CD25⁺ T_reg cells: CD4⁺CD25⁺ T_reg-depletedPBMC) (Fig. 4A, left-hand side panels; Fig. 4B). All 5 CMV-specificCD8⁺ T-cell responses were suppressed by repletion of CD4⁺CD25⁺T_reg cells. (Fig. 4, right-hand side panels). The effect ofadding back CD4⁺CD25⁺ T_reg cells (repletion) could not be overcomeby supplementation with IL-2 (Fig. 4B).

View larger version (30K):
[in this window]
[in a new window]
FIG. 4. Repletion of CD4⁺CD25⁺ T_reg cells to CD4⁺CD25⁺ T_reg cell-depleted PBMC inhibits CD8⁺ T-cell peptide-induced proliferation. Proliferation in response to peptide stimulation was measured by tetramer frequency. Examples are shown with HCV peptide-specific (left-hand panels) or CMV peptide-specific (right-hand panels) expansions. CD4⁺CD25⁺ T_reg-depleted PBMC (top panels), repletion of CD4⁺CD25⁺ T_reg-depleted PBMC with CD4⁺CD25⁺ T_reg cells at 1:10 (middle panels), or 1:2 (CD4⁺CD25⁺ T_reg cell: PBMC) (bottom panels). (B) Group data (n = 12) of HCV-specific tetramer frequency following peptide restimulation with or without the addition of CD4⁺CD25⁺ T_reg cells. Repletion of CD4⁺CD25⁺ T_reg cells was also performed with the addition, on day 0, of IL-2 at concentrations of 200 U/ml and 400 U/ml.

Overall, depletion of CD4⁺CD25⁺ T_reg cells led to a significantincrease in the expansion of HCV-specific CD8⁺ T cells in HCV-infectedindividuals (Fig. 5A). A consistent but less marked increasein CMV- and EBV-specific responses after restimulation was alsoseen in HCV infected patients. No significant effect was seenon either EBV- or CMV-specific CD8⁺ T-cell expansion followingdepletion of CD4⁺CD25⁺ T_reg cells in six healthy individuals(Fig. 5A).

View larger version (16K):
[in this window]
[in a new window]
FIG. 5. Virus-specific responses are suppressed by CD4⁺CD25⁺ T_reg cells in HCV patients compared to healthy individuals. (A) The ratio of the specific CD8⁺ T-cell increase following CD4⁺CD25⁺ T_reg cell depletion over PBMC alone of all patients studied. The EBV- and CMV-specific responses in healthy individuals are in the left column, the EBV- and CMV-specific responses in HCV-infected patients are in the middle column, and the HCV-specific responses in HCV-infected patients are in the right column. The horizontal line in each column represents the median. (B) Depletion of CD4⁺CD25⁺ T_reg cells enhances IFN- ${gamma}$ secretion by virus-specific CD8⁺ T-cell in HCV-infected patients. PBMC alone (PBMC) and following depletion of CD4⁺CD25⁺ T_reg cells (Depleted) were used in an IFN- ${gamma}$ ELIspot assay using a pool of three HCV peptides or (C) the EBV peptide.

CD4⁺CD25⁺ T_reg cells control HCV CD8⁺ T-cell IFN- ${gamma}$ secretion. An ELISpot to quantify the number of IFN- ${gamma}$ -secreting HCV-specificCD8⁺ T cells was used to analyze the effects of CD4⁺CD25⁺ T_regcells in short-term assays. Six patients from the original cohortof 20 were selected such that three had increased HCV tetramerfrequency following depletion of CD4⁺CD25⁺ T_reg cells, while3 did not (negative control) and all six had detectable EBVresponses. Increased numbers of IFN- ${gamma}$ ⁺ HCV-specific CD8⁺ T cellswere seen with 3 pooled HCV peptides following CD4⁺CD25⁺ T_regcell depletion in all three responders (Fig. 5B). IFN- ${gamma}$ ⁺ HCV-specificCD8⁺ T cells were not detected in nonresponders pre- or post-CD4⁺CD25⁺T_reg cell depletion. However, increased numbers of IFN- ${gamma}$ ⁺ EBV-specificCD8⁺ T cells were seen following CD4⁺CD25⁺ T_reg cell depletionin all 6 patients (Fig. 5C).

HCV-specific CD8⁺ T cells ex vivo do not express TGF-ß and IL-10 receptors. Activated CD4⁺CD25⁺ T_reg cells express membranous TGF-ßand secrete IL-10 (6, 14, 25). TGF-ß and IL-10 receptorexpression on virus-specific CD8⁺ T cells was therefore determined.HCV-specific CD8⁺ T cells (n = 4) revealed no expression ofTGF-ß and IL-10 receptors (Fig. 6 A and B). Similarly,CMV-specific CD8⁺ T cells revealed little expression of eitherthe TGF-ß receptor (Fig. 6C) or IL-10 receptor exvivo (Fig. 6D). Expression of both receptors on tetramer-positivecells was observed, however, after in vitro peptide stimulation(Fig. 6E and F). CMV- and EBV-specific CD8⁺ T cells from healthyindividuals had similar levels of IL-10 and TGF-ßreceptor expression ex vivo (i.e., <3%; n = 3) which, alsoincreased after peptide stimulation (data not shown).

View larger version (29K):
[in this window]
[in a new window]
FIG. 6. Expression of the TGF-ß receptor type II and IL-10 receptors on virus-specific CD8⁺ T cells. (A) TGF-ß receptor type II and (B) IL-10 receptor expression on HCV-specific CD8⁺ T cells ex vivo. (C) TGF-ß receptor type II and (D) IL-10 receptor expression on CMV-specific CD8⁺ T cells ex vivo. (E) TGF-ß receptor type II and (F) IL-10 receptor expression on CMV-specific CD8⁺ T cells after peptide restimulation.

DISCUSSION

Top

Discussion

Hepatitis C is a major pandemic that affects more than 170 millionpeople worldwide. Persistent infection is associated with ineffectiveCD4⁺ and CD8⁺ T-cell responses. The causes of T-cell failureare unclear but may include viral variation, T-cell exhaustion,dysfunction of dendritic cells, modulation by specific viralproteins such as core, and intrahepatic death of T cells (6,40). Another hallmark of the poor response is lack of CD8⁺ T-cellmaturation despite initial priming and continued antigen exposure(4, 16). We proposed that CD4⁺CD25⁺ T_reg cells, which regulatehuman and mouse CD4⁺ and CD8⁺ T-cell function, could modulateHCV-specific CD8⁺ T-cell responses.

CD4⁺CD25⁺ T_reg cells isolated from patients with persistentHCV infection were hypo-proliferative and expressed intracellularCTLA-4. They inhibited anti-CD3 stimulated CD4⁺CD25^– proliferation,anti-CD3/anti-CD28 CD8⁺ T-cell proliferation, and HCV peptide-drivenT-cell proliferation, consistent with functional human and murinestudies of the CD4⁺CD25⁺ T_reg cell subset. CD4⁺CD25⁺ T_reg cellsalso inhibited acquisition of CD8⁺ T-cell perforin and IFN- ${gamma}$ expression.

The CD4⁺CD25⁺ T_reg:CD8⁺ T-cell ratio used in these cocultureexperiments was high considering ratios of cells in peripheralblood. However, the ratio of CD4⁺CD25⁺ T_reg cells to CD8⁺ Tcells, as we show, may be relatively high, and particularlywithin portal tracts, where their effects may be relevant. Thepercentage of CD4⁺CD25⁺ T_reg cells within the liver ( ${approx}$ 9%) differssignificantly from that of another recent study (7). However,the percentage within this study was determined in livers atthe time of transplantation, which indicates that these liversmay be cirrhotic and thus differ from livers studied at earlierstages of disease.

The mechanism of action of CD4⁺CD25⁺ T_reg cells was not addresseddirectly. It is thought that TGF-ß and IL-10 playimportant roles in CD4⁺CD25⁺ T_reg cell control in autoimmunedisease (5, 28, 32). Circulating HCV-specific CD8⁺ T cells exhibitno expression of TGF-ß and IL-10 receptors, and onlyweak expression was seen on EBV- or CMV-specific CD8⁺ T cells.However, both receptors were readily up-regulated upon stimulation,which indicates that TGF-ß and IL-10 may have an alternatesuppressive role on activated CD8⁺ T cells.

To date, in vitro experiments have failed to conclusively definea role for TGF-ß or IL-10 in human CD4⁺CD25⁺ T_regcell function (8). However, this does not exclude a role invivo. A recent study has shown an effect of TGF-ßbut not IL-10 on HCV-specific CD8⁺ T cells during chronic HCVinfection (7). This study identified TGF-ß-secretingantigen-specific CD4⁺ T cells and analyzed the effect of anti-TGF-ßantibody on IFN- ${gamma}$ expression by PBMC. It also showed that thesuppression of proliferation of CD4⁺CD25^– T cells wascontact dependent. Our data, together with those of Boettleret al. (6a), also suggest that the suppression of proliferation(in the case of CD8⁺ T cells) is contact dependent. However,in both of these studies proliferation was not inhibited byanti-TGF-ß antibody. These data potentially suggestthat assays of different CD8⁺ T-cell functions may identifydistinct activities of membrane-bound and secreted TGF-ß.Potential differences in activity of CD4⁺CD25⁺ T_reg cells onHCV-specific CD4⁺ and CD8⁺ T cells requires further investigation.

The significant increase in proliferation for all virus-specificCD8⁺ T cells in the absence of CD4⁺CD25⁺ T_reg cells in patientswith chronic HCV infection suggests that these cells are capableof controlling expansion of CD8⁺ T cells during HCV infection.Furthermore when CD4⁺CD25⁺ T_reg cells were added back to depletedPBMCs, proliferation was reduced. These findings are consistentwith a murine study in which depletion of CD4⁺CD25⁺ T_reg cellsenhanced secondary proliferation of CD8⁺ T-cell memory responses(30). In addition, we have shown that the suppression is notdue to removal of IL-2 by CD4⁺CD25⁺ T_reg cells as has previouslybeen suggested (10). High doses of IL-2 added at the time ofpeptide stimulation or thereafter had no effect on the lackof proliferation seen in the repletion experiments and, togetherwith the cell-cell contact dependent mechanism of inhibition,indicate that IL-2 has no role in suppression.

The finding that the depletion of CD4⁺CD25⁺ T_reg cells in patientswith chronic HCV also enhanced expansion of EBV- and CMV-specificCD8⁺ T cells was not mirrored in healthy controls. This maysuggest that CD4⁺CD25⁺ T_reg cells are more active in patientswith chronic HCV and have effects on non-HCV-specific CD8⁺ Tcells. These data are also consistent with recent evidence ofinhibition of maturation of CMV-specific CD8⁺ T cells in persistentHCV infection (27). Our own and previous published data havealso indicated that CD8⁺ T-cell maturation is inhibited in chronicHCV infection, as measured by reduced perforin expression (27,31).

Therefore, a proposed mechanism by which suppression of proliferationand differentiation of CD8⁺ T cells during HCV infection occursat the immature central memory T-cell phase could contributeto the apparent immaturity of CD8⁺ T cells in addition to thelow frequency of HCV-specific CD8⁺ T cells. The frequency ofother virus-specific CD8⁺ T-cell responses (such as CMV) hasnot been shown to alter during HCV infection (18, 34), althoughthese populations are very variable between individuals andmay change with age (21). It is likely that such T-cell populationsare established prior to HCV infection. Thus, the overall consequencesmay be an impact of HCV persistence on the maturation stateof CMV and bulk CD8⁺ T cells rather than a decrease in the precursorpool. Additionally, the suppressive effect on EBV- and CMV-specificCD8⁺ T-cell responses was not as high as the effect on the HCVresponse (Fig. 5A), which may allow those former populationsto maintain a relatively higher frequency.

These data confirm and extend recent studies of the role ofCD4⁺CD25⁺ T_reg cells in chronic human virus infections. Onestudy of HCV, based upon depletion of CD4⁺ T cells, revealedincreased numbers of IFN- ${gamma}$ -secreting cells (38). A further studyin human immunodeficiency virus infection also suggested CD4⁺CD25⁺T_reg cell activity in chronic infection (1). Neither of thesestudies addressed the influence of such cells on proliferationand maturation. The inhibitory effect of CD4⁺CD25⁺ T_reg cellson CD8⁺ T-cell expansion and function may be one mechanism wherebychronic HCV infection is associated with limited CD8⁺ T-cellexpansion and maturation. It may represent a direct effect ofthe virus or be part of the normal response to chronic inflammation,either hepatic or systemic.

Our results complement and are reinforced by another group (6a)that used CD4⁺ depleted PBMC to show the same suppression ofvirus-specific CD8⁺ T-cell proliferation in both chronic andresolved HCV patients but not in healthy individuals. Theirresults also show similar suppression of virus-specific CD8⁺T-cell proliferation (HCV- and influenza virus-specific responses,compared to our HCV-, EBV- and CMV-specific responses) and werealso able to demonstrate that this effect was cell-contact dependentand showed similar potency in titration experiments. Similarly,they were also able to show that this effect was TGF-ßand IL-10 independent.

These results provide a plausible explanation for the low frequenciesand retarded maturation state (low perforin and IFN- ${gamma}$ expression)of HCV-specific CD8⁺ T cells seen in persistently infected patientsand indicate that modulation of CD4⁺CD25⁺ T_reg cell functionmay be one therapeutic strategy for the treatment of chronicHCV.

ACKNOWLEDGMENTS

This study was supported by the Digestive Disorders Foundation(S.M.R. and G.J.M.A.), the Wellcome Trust (S.M.W. and P.K.),and EU grant QLK2-CT-1999-00356 (M.L.).

FOOTNOTES

* Corresponding author. Mailing address: Peter Medawar Building for Pathogen Research, Nuffield Department of Clinical Medicine, University of Oxford, South Parks Road, Oxford OX1 3SY, United Kingdom Phone: 44 1865 281885. Fax: 44 1865 281890. E-mail: paul.klenerman{at}medawar.ox.ac.uk.

These authors contributed equally to this work.

Present address: Centre for Clinical Immunology and BiomedicalStatistics, Murdoch University, Second Floor, North Block, RoyalPerth Hospital, Perth 6000, Australia.

REFERENCES

Top