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The Journal of Immunology, 2007, 179, 1039-1048
Copyright © 2007 by The American Association of Immunologists, Inc.
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Parallel Expansion of Human Virus-Specific FoxP3– Effector Memory and De Novo-Generated FoxP3+ Regulatory CD8+ T Cells upon Antigen Recognition In Vitro1

Eva Billerbeck, Hubert E. Blum and Robert Thimme2

Department of Medicine II, University Hospital Freiburg, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although FoxP3 has been shown to be the most specific marker for regulatory CD4+ T cells, its significance in the CD8+ T cell population is not well understood. In this study, we show that the in vitro stimulation of human PBMC with hepatitis C virus or Flu virus-specific peptides gives rise to two distinct Ag-specific T cell populations: FoxP3– and FoxP3+CD8+ T cells. The FoxP3+ virus-specific CD8+ T cells share phenotypical markers of regulatory T cells, such as CTLA-4 and glucocorticoid-induced TNFR family-related gene, and do produce moderate amounts of IFN-{gamma} but not IL-2 or IL-10. IL-2 and IL-10 are critical cytokines, however, because the expansion of virus-specific FoxP3+CD8+ T cells is blocked by IL-2- or IL-10-neutralizing mAbs. The virus-specific FoxP3+CD8+ T cells have a reduced proliferative capacity, indicating anergy, and display a cell-cell contact-dependent suppressive activity. Taken together, our results indicate that stimulation with a defined viral Ag leads to the expansion of two different cell populations: FoxP3– memory/effector as well as FoxP3+ regulatory virus-specific CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells are critical mediators of dominant immune tolerance to self and foreign Ags. The lineage-specific transcription factor FoxP3 is believed to be the most specific marker for natural CD4+ regulatory T cells (1) that can develop intra- and extrathymically (2, 3). It is generally accepted that CD4+ regulatory T cells are responsible for the control of autoreactive T cells. A growing number of studies suggest that regulatory T cells also play a significant role in down-regulating the immune response to a variety of nonself Ags, such as tumor Ags or microbial Ags, e.g., hepatitis C virus (HCV)3 (4, 5).

The biological role of CD8+ regulatory T cells is less understood, although there is increasing evidence that different subsets of CD8+ T cells possess a regulatory ability in humans and mice (6, 7, 8, 9, 10, 11, 12, 13). Of note, FoxP3+CD8+ T cells have not been identified in a significant amount ex vivo in humans. Upon in vitro stimulation with anti-CD3, however, a small subset of human CD8+ T cells has been shown to transiently up-regulate FoxP3 (14). These induced FoxP3+CD8+ T cells did not display other typical phenotypical markers of regulatory T cells and did not suppress Th1 cytokine expression (14). MHC class I-restricted FoxP3+CD8+CD25+ T cells have been identified after repeated stimulation of human CD8+ T cells with LPS-activated dendritic cells, followed by cloning. These CD8+ T cell clones were not anergic, produced IL-4 and IL-13 but not IFN-{gamma}, and displayed a cell-cell contact-dependent suppressive activity (15).

The origin of FoxP3+CD8+ T cells in humans is not entirely clear. It is possible that pre-existing FoxP3+ regulatory T cells are recruited to the sites of active immune responses where they suppress Ag-specific immune responses. This would, for example, explain why FoxP3+CD8+ T cells are rarely seen in peripheral blood. However, it is also possible that FoxP3– T cells are activated in a way to give rise to an Ag-specific population consisting of both effector and regulatory FoxP3+ T cells (16, 17).

To address this important question, we analyzed FoxP3 expression in HCV- and Flu-specific CD8+ T cells in chronically HCV-infected patients and healthy subjects ex vivo and after peptide-specific stimulation. The ability to analyze FoxP3 expression at the single-cell level by intracellular staining with FoxP3-specific Abs represents a major advance that allows the identification of Ag-specific FoxP3+CD8+ T cells and their phenotypical and functional characterization. Importantly, we found that the in vitro stimulation of human PBMC with HCV- or Flu-specific peptides gives rise to two distinct Ag-specific T cell populations: FoxP3– and FoxP3+CD8+ T cells. The virus-specific FoxP3+CD8+ T cells display some typical phenotypical markers of regulatory T cells, have a reduced proliferative capacity, indicating anergy, and display a cell-cell contact-dependent suppressive activity. IL-2 and IL-10 are critical cytokines, because the addition of neutralizing mAbs significantly reduces the generation of FoxP3+CD8+ T cells. These findings have important therapeutic implications.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Subjects

Blood samples were obtained from eight HLA-A2-positive chronically HCV-infected patients and two HLA-A2-positive healthy blood donors after informed consent and in agreement with federal guidelines and A2 local ethics committee. Liver biopsies were obtained from two HLA-A2-positive patients. The subjects’ characteristics are summarized in Table I. All experiments described below were performed in duplicate.


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  		Table I. Subject characteristics

 
PBMC

PBMC were isolated from EDTA blood by a Ficoll-Histopaque density gradient centrifugation (Pancoll; Pan Biotech). Isolated cells were washed twice in PBS (Invitrogen Life Technologies) and either analyzed immediately or cryopreserved in medium containing 80% FCS (Invitrogen Life Technologies), 10% RPMI 1640 (Invitrogen Life Technologies), and 10% DMSO (Sigma-Aldrich).

Peptides and HLA-A2 tetramers

HCV- and influenza virus-derived peptides previously shown to be HLA-A2-restricted epitopes were purchased from Biosynthan. The amino acid sequences of the HLA-A2-restricted HCV and influenza virus epitopes are as follows: ALYDVVTKL for HCV NS5B 2594, CINGVCWTV for HCV NS3 1073, and GILGFVFTL for Influenza Matrix 58. HLA-A2 tetramers corresponding to the HCV peptides were provided by the National Institutes of Health Tetramer Facility (Atlanta, GA) for HCV and ProImmune for influenza virus.

Antibodies

Anti-CD8-PerCP, anti-CD8-PE, anti-CD4-allophycocyanin, anti-IFN-{gamma}-FITC, anti-IL-2-FITC, anti-CD38-FITC, anti-CD45RA-FITC, anti-CTLA4-allophycocyanin, anti-Bcl-2-FITC, anti-granyzme B-FITC, isotype PE, isotype FITC, and isotype allophycocyanin were obtained from BD Pharmingen. Anti-CTLA-4-FITC, anti-CD122-allophycocyanin, anti-glucocorticoid-induced TNFR family-related gene (GITR)-FITC, and anti-CCR7-FITC were obtained from R&D Systems. Anti-FoxP3-PE, anti-FoxP3-allophycocyanin, anti-perforin-FITC, and anti-CD103-FITC were purchased from eBioscience. Anti-CD127-PE was purchased from Immunotech and anti-CD25-FITC was obtained from Diaclone.

Isolation/depletion of lymphocyte subsets

The MidiMACS separation system (Miltenyi Biotec) was used for the isolation or depletion of lymphocyte subsets according to the manufacturer’s instructions. For the isolation of Ag-specific CD8+ T cells, peptide-stimulated PBMC were stained with the corresponding allophycocyanin-conjugated HLA-A2 tetramer and then incubated with allophycocyanin-conjugated microbeads.

Ag-specific T cell proliferation

A total of 2 x 106 PBMC was resuspended in 1 ml of RPMI 1640 (Invitrogen Life Technologies) containing 10% FCS, 1% streptomycin-penicillin, and 1.5% HEPES buffer (1 M), stimulated with 10 µg/ml synthetic HCV or influenza virus peptide and 0.5 µg/ml anti-human CD28 (BD Pharmingen), and cultured in a 48-well plate (Greiner) at 37°C with 5% CO2. On day 4, cells were stimulated with 0–2000 U/ml human rIL-2 (Hoffmann La Roche). After 7 days of incubation, cells were analyzed for Ag-specific responses or restimulated with 10 µg/ml peptide and 1 x 106 autologous irradiated PBMC (30 Gy). Restimulated cells were analyzed for Ag-specific responses on day 14 after a second stimulation with 0–2000 U/ml human rIL-2 on day 10.

Tetramer and Ab staining

These assays were performed as previously described (18, 19). Briefly, 0.5 x 106 cells per well on a 96-well plate (Greiner) were incubated with allophycocyanin-conjugated HLA-A2 tetramers at 37°C with 5% CO2 for 10 min. Cells were washed three times with PBS containing 1% FCS, blocked with pure IgG1 (BD Pharmingen) for 15 min, stained with surface Abs for 20 min, and washed again three times with PBS containing 1% FCS.

Intracellular Ab and cytokine staining

For intracellular FoxP3 staining, 0.5 x 106 cells per well on a 96-well plate were stained with HLA-A2 tetramers and surface Abs as described above followed by FoxP3 staining using the eBioscience FoxP3 staining buffer set according to the manufacturer’s instructions. Costaining of FoxP3 and intracellular Bcl-2, CLTA-4, GITR, granzyme B, or perforin was done in the same way. For intracellular costaining of FoxP3 and IFN-{gamma} or IL-2, 0.2 x 106 cells per well on a 96-well plate were stimulated with peptides (10 µg/ml) or PMA in the presence of 50 U/ml human rIL-2 and 1 µl/ml Golgiplug (BD Pharmingen). After 5 h of incubation at 37°C with 5% CO2, cells were blocked, stained with anti-CD8 Ab, and washed three times. Cells were stained for intracellular FoxP3 and IFN-{gamma} or IL-2 using the eBioscience FoxP3 staining buffer set. For FACS analysis, all stained cells were fixed in 2% paraformaldehyde. FACS analysis was performed using a BD FACSCalibur or a BD FACSCanto II flow cytometer and FlowJo software (Tree Star).

Ex vivo staining of intrahepatic T cells

For ex vivo staining of intrahepatic lymphocytes, the cell suspension from homogenized liver biopsies was stained in one well on a 96-well plate with a pool of HCV-specific tetramers, followed by cell surface staining and intracellular FoxP3 staining as described above.

CFSE

A total of 2 x 106 cells was incubated with 5 µM CFSE (Molecular Probes) for 10 min at 37°C. After incubation, cells were washed three times with complete medium. Cells were labeled with CFSE either before peptide stimulation for 7 days or before peptide restimulation for an additional 7 days.

Suppression and cytokine neutralizing assays

After 14 days of Ag-specific stimulation, tetramer (Tet+)CD8+ T cells were isolated from PBMC by positive selection as described above. PBMC were cultured in the absence of exogenous IL-2 or in the presence of 500 U/ml IL-2 to obtain Tet+CD8+ T cells with different FoxP3+ expression. To assess the suppressive capacity of FoxP3+ cells, Tet+CD8+ T cells with a significant FoxP3 expression after isolation (25–35%) and Tet+CD8+ T cells negative for FoxP3 after isolation were cocultured with 2 x 105 autologous PBMC that were labeled with CFSE and stimulated with 0.04 µg/ml human anti-CD3 (Immunotech) in a ratio of 1:1 for 7 days in a 96-well plate. In Transwell experiments, Tet+CD8+ T cells were added to the upper chamber of a 6.5-mm Transwell plate with a 0.4-µm pore-sized membrane (Corning) as described previously (20). For cytokine neutralizing assays, cells were cultured in the presence of 0.5 µg/ml mouse anti-human IL-10 (R&D Systems) or in the presence of 0.5 µg/ml anti-human IL-2 (R&D Systems). To analyze the proliferation of anti-CD3-stimulated autologous CD4+ and CD8+ T cells in the presence of Tet+CD8+ T cells with different FoxP3 expression, cells were stained with allophycocyanin-conjugated tetramers, anti-CD4 Ab, anti-CD8 Ab, and anti-FoxP3 Ab. To exclude Tet+FoxP3+ T cells from analysis, cells were gated on Tet–FoxP3– T cells before CD8+ or CD4+ T cells were gated. FoxP3+ expression on anti-CD3-stimulated T cells was negligible.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of FoxP3 expression in HCV- and Flu-specific CD8+ T cells after in vitro peptide-specific stimulation

To investigate the level of FoxP3 expression in virus-specific CD8+ T cells, we tested PBMC from chronically HCV-infected patients and healthy blood donors for their FoxP3 expression using class I tetramers specific for HCV- and Flu-specific peptides (Fig. 1). Of note, as shown for patients 3 and 4 as representative subjects with chronic HCV infection in Fig. 1A, no significant FoxP3 expression of virus-specific CD8+ T cells was detectable ex vivo. These results are in agreement with previous reports that have also described negligible numbers of FoxP3+ cells in the CD8+ T cell compartment ex vivo (14). Furthermore, we did not find a significant expression of FoxP3 in subjects 3 and 4 with detectable intrahepatic HCV-specific CD8+ T cell responses (Fig. 1A). Next, we analyzed the FoxP3 expression in HCV and Flu tetramer-positive CD8+ T cells after 1 wk of peptide-specific stimulation in several patients. Of note, as shown in Fig. 1, B and C, a large fraction (8–62%) of peptide-specific CD8+ T cells expressed FoxP3 after 1 wk of in vitro peptide stimulation, suggesting that specific TCR interaction might lead to the induction of FoxP3 in virus-specific CD8+ T cells. FoxP3 expression was detectable in HCV- as well as Flu-specific CD8+ T cells in chronically HCV-infected patients and in Flu-specific CD8+ T cells in healthy blood donors (Fig. 1C). These results suggest that induction of FoxP3 expression is not limited to virus-specific CD8+ T cells targeting a persisting virus or to virus-specific CD8+ T cells present in chronically infected subjects.


Figure 1
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  		FIGURE 1. Expression of FoxP3 is induced in virus-specific CD8+ T cells after in vitro peptide-specific stimulation. A, PBMC and intrahepatic lymphocytes from two HLA-A2-positive chronically HCV-infected subjects were analyzed ex vivo for FoxP3 expression in virus-specific CD8+ T cells by flow cytometry using HCV-specific HLA-A2 class I tetramers and intracellular FoxP3 staining. Cells were stained with allophycocyanin-labeled tetramer, anti-CD8-PerCP, and anti-FoxP3-PE Abs. Plots are gated on CD8+ T cells. B, Representative plots from indicated subjects show the FoxP3 expression in virus-specific CD8+ T cells ex vivo and after 1 wk of HCV- or Flu peptide-specific stimulation in the presence of 20 U/ml IL-2. C, FoxP3 expression in virus-specific CD8+ T cells from eight HLA-A2-positive chronically HCV-infected patients and two HLA-A2-positive healthy blood donors after 1 wk of HCV- or Flu peptide-specific stimulation in the presence of 20 U/ml IL-2. The percentage of Tet+CD8+ T cells are indicated on top of each column. D, PBMC were labeled with 5 µM CFSE and stimulated with HCV- or Flu-specific peptides in the presence of 20 U/ml IL-2. After 1 wk in culture, virus-specific cells were analyzed for proliferative capacity and FoxP3 expression. Representative dot plots for HCV-specific proliferation of subject 2 are shown. The left plot is gated on CD8+ T cells; the right plot is gated on tetramer-positive CD8+ T cells.

 
To further examine FoxP3 induction in vitro, CFSE-labeled PBMC were stimulated by peptides, followed by the determination of the proliferative responses and FoxP3 expression by flow cytometry. Of note, as shown for representative subject 2 in Fig. 1D, after 7 days of in vitro peptide stimulation, FoxP3 was primarily expressed in highly divided tetramer-positive CFSE-low cells. These results show that proliferation is required for the significant increase of virus-specific FoxP3+CD8+ T cells.

Effects of IL-2 and -10 on the generation of FoxP3+ virus-specific CD8+ T cells

To investigate the role of IL-2 and -10 in the generation of virus-specific FoxP3+CD8+ T cells, we examined FoxP3 expression after 1 wk of peptide-specific stimulation in the absence or presence of different amounts of IL-2 and in the presence of IL-2- and IL-10-neutralizing mAbs. Initially, we focused on the role of IL-2 because of its known ability to both stimulate and terminate T cell responses, its important function in FoxP3-expressing regulatory CD4+ T cells and because the addition of 20 U/ml IL-2 on day 3 is part of our protocol that we used to expand virus-specific CD8+ T cells (Fig. 1) (see Materials and Methods) (21, 22). Importantly, as shown in Fig. 2 for individuals 1 and 10 as representative subjects, we found a clear association between the amount of IL-2 and the expression of FoxP3 in peptide-specific induced virus-specific CD8+ T cells tested on day 7. Indeed, in the absence of exogenous IL-2, most virus-specific CD8+ T cells displayed no significant FoxP3 expression. In the presence of IL-2, however, a dose-dependent increase in FoxP3 expression of virus-specific CD8+ T cells was detectable (Fig. 2A). As shown in Fig. 2B, the peptide-specific induction of FoxP3 in virus-specific CD8+ T cells is seen across a wide range of peptide concentrations and decreases with minute amounts of peptides. To further investigate the role of IL-2 and IL-10 in the induction of FoxP3+CD8+ T cells, neutralizing anti-IL-2 or -10 mAb were added to the cultures before peptide-specific stimulation. Importantly, the addition of both mAb resulted in a significantly reduced expansion of virus-specific FoxP3+CD8+ T cells (Fig. 2C). Of note, after the addition of anti-IL-10, we observed in both subjects a significantly better expansion of virus-specific CD8+ T cells that were primarily FoxP3–. For example, in the presence of anti-IL-10, the frequency of virus-specific CD8+ T cell as measured by specific tetramer staining increased from 4.7 to 10% (in the presence of 20 U/ml IL-2) and from 2.3 to 6.0% (in the presence of 500 U/ml IL-2) in subject 1, from 2.1 to 6.3% (in the presence of 20 U/ml IL-2) and from 1.3 to 5.8% (in the presence of 500 U/ml IL-2) in subject 10. These results indicate that IL-10 has at least two different but maybe functionally overlapping biological functions in this setting: First, it is required for the generation of FoxP3+CD8+ T cells. Second, it inhibits the expansion of FoxP3– virus-specific CD8+ T cells. Of note and in contrast to the clear effects observed after the addition of anti-IL-10, we did not find an increased expansion of virus-specific FoxP3+CD8+ T cells after the addition of anti-TFG-beta (data not shown). However, as shown for anti-IL-10, the overall expansion of virus-specific CD8+ T cells was slightly increased in the presence of anti-TGF-beta (data not shown), supporting a recent study that analyzed the effect of TGF-beta on HCV-specific T cells (23).


Figure 2
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  		FIGURE 2. Effects of IL-2 and IL-10 on FoxP3 induction in virus-specific CD8+ T cells. PBMC were stimulated either with HCV- or Flu-specific peptides in the presence of different IL-2 concentrations (A), with different concentrations of HCV or Flu peptides in the presence of 20 U/ml or 500 U/ml IL-2 (B), or with HCV- or Flu-specific peptides, 20 or 500 U/ml IL-2 and IL-10- or IL-2- neutralizing Abs as indicated (C). After 1 wk in culture Tet+CD8+ T cells were analyzed for FoxP3 expression by flow cytometry. Representative results from subject 1 (HCV-specific stimulation) and subject 10 (Flu-specific stimulation) are shown. In C, the percentage of virus-specific CD8+ T cells is indicated on top.

 
FoxP3 expression is specifically induced in virus-specific CD8+ T cells

Next, we determined whether FoxP3+ expression in virus-specific CD8+ T cells is indeed Ag specific. For these experiments, the cells were not only stimulated with the respective virus-specific peptide but also with anti-CD3 alone because it has been recently shown that activation with anti-CD3 can transiently induce FoxP3 expression in CD8+ T cells (14). Specifically, PBMC from all chronically HCV-infected patients and healthy subjects were stimulated either with the respective HCV peptide or anti-CD3 in the presence of different amounts of IL-2 (0, 20, and 500 U/ml). Not surprisingly, as shown for two representative subjects (individuals 2 and 9), stimulation with specific peptides did result in a better expansion of virus-specific CD8+ T cells compared to unspecific stimulation with anti-CD3 (Fig. 3A). Indeed, in the absence of IL-2, virus-specific CD8+ T cells were detectable by tetramer in 1.9% of peptide-specific and in only 0.3% of anti-CD3-stimulated cell lines. The peptide-specific stimulated tetramer-positive CD8+ T cells displayed a higher expression of FoxP3 compared with anti-CD3-stimulated virus-specific CD8+ T cells. As expected from our previous observations, the stimulation was most prominent in the presence of IL-2 (20 or 500 U/ml) where we also observed a better expansion of the virus-specific CD8+ T cells. However, in the absence of specific peptide stimulation, IL-2 alone had only a minor effect on the FoxP3 expression of virus-specific CD8+ T cells and only at a high IL-2 concentration (500 U/ml) (Fig. 3A). It is also important to note that a significant increase of FoxP3 was only detectable in tetramer-positive and not in tetramer-negative CD8+ T cells, even though a similar anti-CD3-induced FoxP3 expression was found in this population (Fig. 3B). Taken together, these results clearly indicate that the induction in FoxP3 expression of virus-specific CD8+ T cells is Ag specific.


Figure 3
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  		FIGURE 3. FoxP3 expression is specifically induced in virus-specific CD8+ T cells. A, PBMC were stimulated with either HCV (subject 2) or Flu (subject 9) peptide or anti-CD3 in the presence of 0, 20, or 500 U/ml IL-2. Selected cultures were stimulated with IL-2 only. Intracellular FoxP3+ expression in Tet+CD8+ T cells was analyzed 1 wk after stimulation. Results show the HCV-specific and Ag-unspecific induction of FoxP3 in Tet+CD8+ T cells from subject 2 and the Flu-specific and Ag-unspecific induction of FoxP3 in Tet+CD8+ T cells from subject 9. The percentage of virus-specific CD8+ T cells is indicated on top. B, FoxP3 expression in the total CD8+ T cell population from subjects 2 and 9 after 1 wk of stimulation.

 
Phenotypical analysis of FoxP3+CD8+ T cells

A phenotypical analysis of the virus-specific FoxP3+CD8+ T cells performed on day 7 revealed that they were largely positive for CTLA-4 and GITR (Fig. 4), which have both been shown to be associated with CD4+CD25+ regulatory T cells (24, 25, 26). By comparison, both markers were only very little expressed in FoxP3–CD8+ T cells (Fig. 4A). We did not find a clear association between CD25 and FoxP3 expression because CD25 was also expressed in FoxP3– cells (Fig. 4). The expression of the activation marker CD38 was also detectable on both CD8+ T cell populations (Fig. 4A). These results indicate that a large fraction of FoxP3+ cells is highly activated. This is not surprising because the cells have been stimulated with a specific peptide and expanded in the presence of IL-2. The virus-specific FoxP3+CD8+ T cells expressed low levels of CD45RA, a classical marker of naive T cells, CCR7 and CD127 (Fig. 4A), thus resembling effector memory CD8+ T cells. These results confirm previous reports that have described this phenotype on peptide-specific expanded virus-specific CD8+ T cells (27, 28). Of note, the FoxP3+CD8+ T cells failed to express significant levels of CD122, which has previously been suggested to be a marker for regulatory CD8+ T cells (9, 29). Interestingly, a larger fraction of virus-specific FoxP3+CD8+ T cells expressed CD103 compared to the FoxP3–CD8+ T cell population. CD103 has previously been shown to be a marker for regulatory CD8+ T cells (30, 31). Finally, we tested these cells for the expression of Bcl-2 and observed that the majority of FoxP3+CD8+ T cells were Bcl-2+ in contrast to the FoxP3– population, suggesting that they are relatively resistant to apoptosis. Of note, the expression of Bcl-2 and resistance to apoptosis has previously been shown to be characteristic for induced regulatory CD8+ T cells (32).


Figure 4
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  		FIGURE 4. Tet+FoxP3+CD8+ T cells differ from Tet+FoxP3–CD8+ T cells in their expression of GITR, CTLA4, and Bcl-2. A, Cell surface expression of CD45RA, CCR7, CD103, CD38, CD25, and intracellular expression of GITR and Bcl-2 of Tet+FoxP3+CD8+ T cells (black lines) and of Tet+FoxP3–CD8+ T cells (gray lines) and the cell surface expression of CD127, CD122 and intracellular expression of CTLA4 of FoxP3+CD8+ (black lines) and FoxP3–CD8+ T cells (gray lines) are shown in representative histograms for subject 1. Cells were stimulated with HCV peptide for 1 wk. B, Expression of GITR, CD25, Bcl-2, and CD103 on Tet+FoxP3+ and Tet+FoxP3–CD8+ T cells and CTLA4 expression on FoxP3+CD8+ and FoxP3–CD8+ T cells from subjects 1, 2, 9, and 10. Every dot represents one patient. T cells were stimulated with either HCV- or Flu-specific peptides for 1 wk before analysis.

 
Functional characterization of FoxP3-expressing virus-specific CD8+ T cells

Next, we determined whether the induction of FoxP3+ expression in virus-specific CD8+ T cells results in functional changes of these cells or whether it just reflects the activation status of in vitro

peptide-stimulated cells. Analysis of intracellular cytokine production has served previously as an indirect way to assess acquisition of regulatory T cell properties by activated T cells (14). To determine whether the induced FoxP3+ cells suppressed IFN-{gamma}, IL-2, or IL-10 production, peptide-specific expanded cells were examined for FoxP3, IL-2, and IFN-{gamma} expression. As shown in Fig. 5A, FoxP3+CD8+ T cells failed to secrete IL-2 after 5 h of peptide-specific stimulation, whereas a significant fraction secreted IFN-{gamma}. FoxP3+CD8+ T cells were also expressing high amounts of granzyme B and perforin (Fig. 5A). No significant IL-10 production was detected in supernatants from cultured peptide-specific CTL lines by ELISA. Furthermore, we did not detect measurable levels of IL-10 by this method (data not shown). Next, we determined the expansion capacity of virus-specific FoxP3+CD8+ T cells in two subjects by restimulating the cells after 1 wk for an additional week. Importantly, we observed that the expansion capacity was highest in the presence of 20 U/ml IL-2 when ~64.5 and 29.9% of CD8+ T cells, respectively, were specific for HCV NS5B after 2 wk of stimulation (Fig. 5B). Of note, a large fraction of these cells was still FoxP3+ after 14 days of stimulation. Peptide-specific cell lines that were restimulated in the presence of 500 U/ml IL-2 also expanded significantly, however, to a lesser extent compared to 20 U/ml IL-2, from 0.2 to 12.3% (subject 1) and from 2.8 to 22.5% (subject 2), respectively. As expected from our previous results, a higher fraction of virus-specific CD8+ T cells expressed FoxP3 in the presence of high concentrations of IL-2. These results suggest that FoxP3 expression is not transient but sustained at least in the presence of ongoing peptide stimulation and high IL-2 concentrations.


Figure 5
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  		FIGURE 5. Functional analysis of virus-specific CD8+ T cells. A, HCV- or Flu peptide-specific T cell lines were restimulated with peptide or PMA and 50 U/ml IL-2. After 6-h incubation, intracellular expression of FoxP3, IL-2, or IFN-{gamma} was analyzed by flow cytometry. Granzyme B and perforin expression in Tet+CD8+ T cells was analyzed after 14 days of Ag-specific stimulation. Representative plots of patient 2 are shown. B, Percentage of virus-specific CD8+ T cells and FoxP3 expression in virus-specific CD8+ T cells was analyzed after 1 and 2 wk of HCV-specific stimulation in the presence of 0, 20, or 500 U/ml IL-2. Results from PBMC of subject 1 and 2 after HCV-specific stimulation are shown. C, PBMC were either labeled with 5 µM CFSE before peptide stimulation for 1 wk or prior peptide restimulation for an additional week. Virus peptide-specific proliferation after HCV-specific stimulation (7 days) or restimulation (14 days) of Tet+FoxP3+ (black lines) and Tet+FoxP3–CD8+ T (gray lines) cells in the presence of 500 U/ml IL-2 is shown in representative plots from subjects 1 and 2. D, Tet+CD8+ T cells were isolated from peptide-specific T cell lines by using allophycocyanin-conjugated beads. Isolated Tet+CD8+ T cells with a FoxP3 expression of 35% (subject 9) and 24% (subject 1) after isolation or Tet+CD8+ T cells negative for FoxP3 after isolation were added in a ratio 1:1 to autologous PBMC that were labeled with CFSE before stimulation with anti-CD3. Selected experiments were performed in the presence of IL-10 neutralization Ab or in Transwell plates. Proliferation of CD8+ and CD4+ T cells was analyzed after 7 days in culture. Representative histograms show the proliferation of CD4+ and CD8+ T cells from subjects 9 and 1.

 
To further determine the proliferative capacity of FoxP3+CD8+ T cells at the single-cell level, peptide-stimulated cells were labeled with CFSE before they were restimulated peptide-specifically for another week. Importantly, as shown in Fig. 5C, we found a significantly reduced proliferative capacity of FoxP3+ compared with FoxP3– virus-specific CD8+ T cells between days 7 and 14. These results suggest that, although proliferation is required for induction of FoxP3 within the first 7 days (in the presence of peptide and IL-2), once induced, FoxP3+CD8+ T cells proliferate only poorly. This may also explain why we failed to generate long-term cultures of virus-specific FoxP3+CD8+ T cells. It is not clear why the FoxP3+ T cells dominate the T cell pool after 2 wk of stimulation despite their reduced proliferation capacity. One explanation may be an accelerated apoptosis rate of FoxP3-virus-specific CD8+ T cells in the presence of high IL-2 concentrations. Indeed, ~50% of cells in the 2-wk culture were annexin positive (data not shown). Because FoxP3 and annexin staining cannot be combined, the T cell subset undergoing apoptosis could not be determined. However, the observation that FoxP3+CD8+ T cells are strongly Bcl-2+, whereas FoxP3– cells are not, suggests that the latter cells are indeed more likely to undergo apoptosis.

Finally, we determined the suppressive capacity of the virus-specific FoxP3+CD8+ T cell population. The intracellular markers FoxP3, GITR, and CTLA-4 cannot be used to isolate virus-specific FoxP3+CD8+ T cells. Thus, to assess the suppressive capacity of Tet+FoxP3+CD8+ T cells, tetramer-positive CD8+ T cells of peptide-stimulated T cell lines with significant FoxP3 expression (24–35%) were purified using magnetic beads and added in a ratio of 1:1 to autologous PBMC, labeled with CFSE before stimulation with anti-CD3. As shown in Fig. 5D, the addition of FoxP3+ tetramer-positive CD8+ T cells with a FoxP3 expression of 24 and 35%, respectively, led to a significant inhibition in the proliferation of anti-CD3-stimulated CD8+ and CD4+ T cells in subjects 1 and 9. By contrast, the addition of tetramer-positive but FoxP3– cells had no significant effect on T cell proliferation (Fig. 5D). The suppressive effect was not abolished by anti-IL-10 but was cell-cell contact dependent because it was reduced by separating the cell suspensions by a Transwell. It is also important to note that tetramer-positive but FoxP3–CD8+ T cells did not display any suppressive activity, further supporting a specific suppressive effect of FoxP3+ virus-specific CD8+ T cells (Fig. 5D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results of our study suggest that the same virus-specific T cell population that is present in the peripheral blood of chronically HCV-infected patients or healthy subjects may give rise to both FoxP3– effector memory and FoxP3+ regulatory T cells upon recognition of the same Ag (Fig. 6). Indeed, our in vitro results indicate that specific peptide recognition and the cytokines IL-2 and IL-10 are required to induce FoxP3 expression in proliferating virus-specific CD8+ T cells. Of note, the de novo generation of virus-specific FoxP3+ T cells has been recently reported for the CD4+ T cell subset. Indeed, Walker et al. (17) used influenza hemagglutinin epitopes to generate hemagglutinin-specific regulatory T cells that required cognate Ag for activation but subsequently suppressed noncognate bystander T cell responses as well. Taken together, these results support the notion that regulatory virus-specific CD4+ and CD8+ T cells can be generated in vitro (6).


Figure 6
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  		FIGURE 6. Model of regulatory CD8+ T cell induction. Virus-specific CD8+ T cells targeting a specific viral Ag can be induced to become FoxP3+ regulatory or FoxP3– effector CD8+ T cells.

 
In a recent study, Gavin et al. (14) reported only a transient up-regulation of FoxP3+ in CD8+ T cells upon in vitro stimulation with anti-CD3. Different from our study, these FoxP3+CD8+ T cells did not change their phenotype or function. Thus, our results suggest that stimulation with virus-specific peptides in the presence of IL-2 induced FoxP3 expression and subsequent regulatory CD8+ T cell development more strongly than polyclonal stimulation with anti-CD3.

The parallel development of effector and regulatory virus-specific CD8+ T cells after Ag recognition may play an important role in T cell homeostasis and viral immunobiology because induced FoxP3+ regulatory CD8+ T cells may limit virus-specific effector mechanisms in a strict Ag-specific manner. Of note, a recent mouse study has described the parallel and sequential development of IL-2-dependent effector and regulatory CD4+ T cells in response to systemic Ag in vivo (33). Indeed, by transfer of naive Ag-specific CD4+ T cells into lymphopenic mice that express an endogenous Ag, the authors observed that T cells responding to this Ag develop into effector cells that cause graft-vs-host disease. Subsequently, however, FoxP3+CD4+ T cells were generated from the same Ag-specific T cell population. Importantly, in the absence of IL-2, no FoxP3+CD4+ T cells were observed. Thus, these in vivo findings in the mouse CD4+ T cell subset share similarities with our results obtained in the human CD8+ T cell subset in vitro and support the concept that T cells have the potential to develop into functionally and phenotypically very different T cell populations after recognition of the same Ag with IL-2 playing a central role in this scenario (Fig. 6).

Of note, however, we were unable to detect virus-specific FoxP3+CD8+ T cells in peripheral blood (HCV and Flu) or HCV-infected livers (HCV) ex vivo. These results indicate that, if the same expansion of FoxP3+CD8+ T cells occurs in vivo, it is not easy to detect, i.e., it may occur primarily in Ag-rich lymph nodes with the appropriate cytokine milieu or during the early phase of acute infection when high amounts of inflammatory cytokines are secreted. Clearly, additional studies are needed to address the role of virus-specific FoxP3+CD8+ T cells during acute viral infection, e.g., acute viral hepatitis to determine whether a similar induction of FoxP3+CD8+ T cells is occurring in vivo. Of note, recently a model has been proposed that links the level of regulatory T cell generation to the extent of immune stimulation (34). Thus, it is well possible that the level of stimulation is too weak during chronic HCV infection to induce FoxP3+ induction. This possibility is supported by previous findings that show a nonactivated central-memory phenotype CD8+ T cell population during chronic HCV infection, at least in the peripheral blood (35, 36).

The induced virus-specific FoxP3+CD8+ T cells display typical phenotypic markers of CD4+ regulatory T cells such as CTLA-4 and GITR, and have suppressive cell-cell contact-dependent activity. Of note, however, we did not find a clear correlation between FoxP3+ and CD25+ or CD122+. Both markers have previously been shown to be associated with certain subsets of CD8+ regulatory T cells (6). In different in vivo or in vitro models, it has been shown that regulatory CD8+ T cells are present or can be induced. For example, Gilliet et al. (10) have reported the IL-10-dependent generation of regulatory CD8+ T cells producing IL-10 and some IFN-{gamma} after in vitro stimulation of naive CD8+ T cells with alloantigen-presenting dendritic cells. Although we also found a clear IL-10 dependence of the induction of FoxP3+CD8+ T cells, those T cells did not produce significant amounts of IL-10. Different from our findings, most studies of regulatory CD8+ T cells reported IL-10 production of this cell population (9, 10, 11, 30, 37, 38). For example, in one study IL-10-producing HCV-specific CD8+ T cells with a regulatory phenotype were found in the liver, although FoxP3 expression was not determined in that study (39). In contrast, similar to our findings, in some studies no significant IL-10 production of regulatory CD8+ T cells could be detected (15, 32, 40). For example, autoreactive human peripheral FoxP3+CD8+ T cell clones with a regulatory function were unable to secrete IL-10 (15). The virus-induced FoxP3+CD8+ T cells in our study were able to produce some IFN-{gamma}, perforin, and granzyme B, as has also been shown for other regulatory CD8+ T cells (11, 14, 37, 40). Of note, perforin and granzyme B have both been shown to be involved in the cell-cell contact-dependent suppression of CD4+ regulatory T cells (41, 42). Taken together, the results suggest that in different models and diseases different subsets of regulatory CD8+ T cells can be induced. This is very similar to the heterogeneous population of regulatory T cells described for the regulatory CD4+ T cell subtype (6).

The origin of the FoxP3+CD8+ T cells could not be determined in our study. It is possible that virus-specific FoxP3+CD8+ cells arise by activation and de novo expression of FoxP3, as has been reported for CD4+ regulatory T cells (2, 17). However, it is also possible that an extensive in vitro expansion of a pre-existing population of Ag-specific FoxP3+CD8+ T cells occurs that is not detectable ex vivo. Another important question that emerges from this finding is whether the regulatory T cells arise from effector cells or whether there are parallel differentiation pathways. Of note, both populations are derived from the CD45RO+ compartment because the in vitro depletion of CD45R0 abolishes the expansion of both cell populations (data not shown). Thus, these results suggest that both cell populations arise from a memory T cell population that has been primed in vivo.

Another interesting finding in our study is the increased frequency of HCV-specific CD8+ T cells that are primarily FoxP3– after the blockade of IL-10 (Fig. 2). These results suggest that IL-10 is not only required for the generation of virus-specific FoxP3+ T cells but that it also has a suppressive effect on the proliferation of virus-specific FoxP3–CD8+ T cells. These data are consistent with two recent reports demonstrating in the lymphocytic choriomeningitis virus mouse model that IL-10 is a single key molecule that directly induces suppression of virus-specific CD8+ T cells (43, 44). Of note, genetic removal or in vivo blockade of the IL-10R with a neutralizing Ab resulted in rapid resolution of a persistent infection and strong and functional virus-specific CD8+ T cell responses (43, 44). Taken together, these results suggest that therapeutic blockade of the IL-10R may result in T cell recovery and prevention of viral persistence.

Our results have further implications because the in vitro expansion of T cells often is an important prerequisite for therapeutical adoptive cell transfer. Importantly, our results indicate that IL-2 may not only promote the expansion of Ag-specific effector but also of regulatory CD8+ T cells that may impair the in vivo activity of adoptively transferred CD8+ T cells. In contrast, the Ag-specific induction of regulatory CD8+ T cells may also be used for the fine-tuning of Ag-specific suppression in various clinical settings. Thus, the ability to manipulate the balance between Ag-specific effector and regulatory T cells by the IL-2 concentration may be a novel therapeutic strategy in several clinical settings.


   Acknowledgments
 
We thank the patients and healthy blood donors for participating in the study after informed consent and in agreement with federal guidelines and the local ethics committee. We thank Dr. Stefan Martin for helpful discussion. The HLA-A2 tetramers were kindly provided by the Tetramer Facility, National Institutes of Health.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft SFB 620, C6 (to R.T.). Back

2 Address correspondence and reprint requests to Dr. Robert Thimme, Department of Medicine II, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany. E-mail address: thimme{at}med1.ukl.uni-freiburg.de Back

3 Abbreviations used in this paper: HCV, hepatitis C virus; GITR, glucocorticoid-induced TNFR family-related gene; Tet, tetramer. Back

Received for publication January 26, 2007. Accepted for publication May 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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