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Keywords:

  • Forkhead box P3;
  • interleukin-10;
  • ribavirin;
  • regulatory T1 cell;
  • regulatory T cell

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Because regulatory T (Treg) cells play an important role in modulating the immune system response against both endogenous and exogenous antigens, their control is critical to establish immunotherapy against autoimmune disorders, chronic viral infections and tumours. Ribavirin (RBV), an antiviral reagent used with interferon, is known to polarize the T helper (Th) 1/2 cell balance toward Th1 cells. Although the immunoregulatory mechanisms of RBV are not fully understood, it has been expected that RBV would affect T reg cells to modulate the Th1/2 cell balance. To confirm this hypothesis, we investigated whether RBV modulates the inhibitory activity of human peripheral CD4+ CD25+ CD127 T cells in vitro. CD4+ CD25+ CD127 T cells pre-incubated with RBV lose their ability to inhibit the proliferation of CD4+ CD25 T cells. Expression of Forkhead box P3 (FOXP3) in CD4+ CD25 T cells was down-modulated when they were incubated with CD4+ CD25+ CD127 T cells pre-incubated with RBV without down-modulating CD45RO on their surface. In addition, transwell assays and cytokine-neutralizing assays revealed that this effect depended mainly on the inhibition of interleukin-10 (IL-10) produced from CD4+ CD25+ CD127 T cells. These results indicated that RBV might inhibit the conversion of CD4+ CD25 FOXP3 naive T cells into CD4+ CD25+ FOXP3+ adaptive Treg cells by down-modulating the IL-10-producing Treg 1 cells to prevent these effector T cells from entering anergy and to maintain Th1 cell activity. Taken together, our findings suggest that RBV would be useful for both elimination of long-term viral infections such as hepatitis C virus infection and for up-regulation of tumour-specific cellular immune responses to prevent carcinogenesis, especially hepatocellular carcinoma.


Abbreviations
COX-2

cyclooxygenase-2

CPA

cyclophosphamide

FOXP3

forkhead box P3

GITR

glucocorticoid-induced tumour necrosis factor receptor

HCV

hepatitis C virus

ICOS

inducible co-stimulatory molecule

IFN

interferon

IL

interleukin

mAb

monoclonal antibody

pB

plate bound

PBMC

peripheral blood mononuclear cell

PE

phycoerythrin

PI

propidium iodide

RBV

ribavirin

TAA

tumour-associated antigen

TGF

transforming growth factor

Th

T helper cells

Th3

T helper type 3 cell

Treg

regulatory T cell

Tregadapt

adaptive regulatory T cell

Tregnat

naturally occurring regulatory T cell

Treg1

regulatory T1 cell

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Specific regulation of the immune system is the ultimate goal for the establishment of therapies against diseases associated with inappropriate immune responses such as autoimmune disorders, chronic viral infections and tumours. Previous reports described the mechanisms of immunological impairments in such diseases,[1-4] and impairment of cellular immune responses is considered to be critical for establishing continuous viral infection[3] or tumour progression.[4] Hence, improvement of antigen-specific cellular immune responses will be essential for establishing immune therapies against these diseases as well as humoral or innate immune responses.[5, 6]

It is well known that cellular and humoral immune responses are regulated through a complicated cascade.[7] Among the elements of that cascade, the T helper (Th) 1/2 cell balance is considered essential to regulate the cellular/humoral immune response.[8, 9] In addition, regulatory T (Treg) cells are also critical for immune regulation.[10] Treg cells have unique characteristics represented by a lack of response to various antigens and the ability to induce Th cells to enter antigen-specific anergy,[10, 11] and human Treg cells exhibit their inhibitory activity through various pathways.[12] Recently, it has been clarified that Treg cells consist of various subsets[13] including naturally occurring Treg (Tregnat) cells that differentiate in the thymus and exhibit inhibitory ability in a cell contact-dependent manner,[14] and adaptive Treg (Tregadapt) cells that differentiate from naive CD4+ T cells under the influence of Tregnat cells[15, 16] and exhibit inhibitory activity in a humoral element-dependent manner.[17, 18] Although Treg cells were first identified as regulators of autoreactive T cells, Treg cells also induce immune responses against exogenous antigens such as acute or chronic infectious viruses[19, 20] or other endogenous antigens such as tumours.[21, 22] The Treg cells can down-modulate antigen-specific Th1 activity in the later phase of viral infections, which in turn switches the dominant immune response from cellular to humoral.[23] In contrast, over-activation of Treg cells would be the principal reason for the impaired cellular immune response in persistent viral infection, such as with hepatitis C virus (HCV).[24] Hence, regulation of Treg cells may improve impaired cellular immune responses against many endogenous and exogenous antigens.

Ribavirin (RBV), a purine nucleotide analogue used as an antiviral reagent,[25] is well known for its contribution to HCV elimination in combination with interferon (IFN).[26] Among the putative mechanisms for the enhancement of viral elimination by RBV, it is notable that RBV polarizes the Th cell balance into Th1 cell dominance.[27-29] Some reports indicated that RBV may maintain Th1 cell activity through inhibition of immunosuppressive cytokines such as interleukin-4 (IL-4)[28] or IL-10,[29] but the immunological mechanisms by which RBV alters the Th1/Th2 balance to Th1 dominance is not fully understood.

Recently, we have demonstrated that RBV down-modulates inducible co-stimulator (ICOS) on human CD4+ T cells, which in turn decreases IL-10 secretion, leading to the maintenance of Th1 activity,[30] and speculated that RBV might affect Treg cells that also express ICOS on their surface. In the present study, we examined the effects of RBV against human peripheral Treg cells in vitro and found the unique characteristics of RBV, which might down-modulate the activity of Treg cells by inhibiting the differentiation of naive CD4+ T cells into Tregadapt cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Cell preparation

Peripheral blood was obtained from five healthy individuals who were serologically confirmed to be free from hepatitis B virus, HCV, or human immunodeficiency virus infection. This study protocol conformed to the ethical guidelines of the Declaration of Helsinki as reflected in a priori approval by the Institutional Review Committee of Nippon Medical School. CD4+ T cells were purified from peripheral blood mononuclear cells (PBMCs) isolated from heparinized blood using the Ficoll–Paque (Amersham, Buckinghamshire, UK) density-gradient method with a magnetic cell sorter (Miltenyi Biotech, Auburn, CA). Briefly, PBMCs were incubated with a CD4+ T-cell isolation cocktail containing biotin-conjugated anti-human CD8, CD14, CD16, CD19, CD36, CD56, CD123, T-cell receptor-γδ, and glycophorin A antibodies (Miltenyi Biotech) for 10 min at 4° and additionally labelled with magnetic bead-conjugated streptavidin for 15 min at 4°. Cells were washed, subjected to LS separation columns, and the pass-through fraction was collected as CD4+ T cells. Because Treg cells could be identified by their CD127 deficiency,[31] CD4+ T cells were subsequently divided into CD25 and CD25+ CD127 cell fractions using FACSort. Briefly, CD4+ T cells were stained with FITC-conjugated anti-human CD25 (BD-Bioscience, San Diego, CA) and Alexa-Fluor647-conjugated anti-human CD127 monoclonal antibodies (mAbs) (BD Bioscience). Cells were sorted into FACS AriAll (BD Bioscience) and both CD25 and CD25+ CD127 cells were collected.

Cell culture reagents

All cells were cultured in complete T-cell medium, RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, HEPES-buffer solution 5 mm, penicillin 100 U/ml, streptomycin100 μg/ml, l-glutamine 2 mm, sodium pyruvate solution 2 mm, and non-essential amino acid solution 2 mm (all these supplements were purchased from Gibco-BRL, Santa Clara, CA), modified vitamins 2 mm (Dainippon Pharmaceutical Co. Ltd., Tokyo, Japan), and 2-mercaptoethanol 2 mm (Sigma Chemical Company, St Louis, MO).

Monoclonal antibodies

Anti-human IL-10 and anti-human transforming growth factor-β1 (TGF-β1) mAbs (e-Bioscience, San Diego, CA) were used for cytokine-neutralizing assays.

Cell stimulation

The isolated CD4+ CD25 and CD4+ CD25+ CD127 T cells were stimulated in vitro with plate-bound (pB) or soluble anti-CD3 antibody (OKT3; e-Bioscience) for analysing cell surface and intracellular molecule expression, cell proliferation and cytokine profiling. RBV 0–500 ng/ml[32] (Sigma Chemicals) reconstructed in PBS was added to the culture plates.

Flow cytometry

Flow cytometric analysis was performed using a FACS Diva (BD Bioscience). For staining cell surface molecules, 500 000 cells were harvested, washed twice with RPMI-1640, and pelleted. The following antibodies were used: FITC-conjugated anti-human CD25 and ICOS, phycoerythrin (PE)-conjugated anti-human CD4, PE-Cy7-conjugated anti-human CD45RO, allophycocyanin-conjugated anti-human CD45RA (all antibodies were purchased from BD Bioscience). The expression of intracellular Forkhead box P3 (FOXP3) was detected using a PE-conjugated anti-human FOXP3 staining kit (e-Bioscience) according to the manufacturer's instructions. Propidium iodide (PI) was used to confirm the percentage of dead cells.

Cytokine assays

CD4+ CD25 and CD4+ CD25+ CD127 T cells were plated at 1 × 106/ml in a 48-well plate and stimulated with pB-OKT3 5·0 μg/ml with or without RBV for 48 hr at 37°. Culture supernatants were collected and stored immediately at 80°. Enzyme-linked immunosorbent assays were performed to titrate IL-4, IL-10, IFN-γ and TGF-β1 in the culture supernatants using DUOSET anti-human IL-4, IL-10, IFN-γ and TGF-β1 ELISA kits (R&D Systems, Minneapolis, MN).

Cell proliferation assay

The [3H]thymidine incorporation assay was performed to determine the impact of RBV on the regulatory effect of CD4+ CD25+ CD127 T cells. Twenty thousand CD4+ CD25 T cells and CD4+ CD25+ CD127 T cells with or without pre-incubation with RBV were mixed and stimulated with pB-OKT3 0·05–5·0 μg/ml in the presence of 2·0 × 105 allogeneic irradiated (3000 rads) PBMCs for 3–7 days at 37° in 96-well round-bottomed culture plates. Subsequently, 1 μCi/well of [3H]thymidine (MP Biomedicals, Morgan City, CA) was added and incubated for an additional 16 hr. The cells were harvested and [3H]thymidine incorporation was measured using a 1450 Micro Beta Trilux scintillation spectrometer (Wallac, Gaithersburg, MD). For cytokine-neutralizing assays, either anti-human IL-10 mAb 1·0 μg/ml or anti-human TGF-β1 mAb 10 μg/ml was added to each culture well. To confirm the regulatory activity of the CD4+ T cells after incubation with CD4+ CD25+ CD127 T cells, whole cells including CD4+ CD25 T cells and CD4+ CD25+ CD127 T cells or those pre-treated with RBV were harvested. Twenty thousand of these cells and the same number of freshly isolated CD4+ CD25 T cells from the same donors were mixed and re-stimulated with pB-OKT3 0·05 μg/ml in the presence of 2·0 × 105 allogeneic irradiated PBMCs for 7 days at 37°. The thymidine incorporation was measured as described above.

Transwell assays

Transwell systems were used to determine the participation of humoral elements in the regulatory effects of CD4+ CD25+ CD127 T cells.[33] Twenty thousand CD4+ CD25 T cells were plated with 1 × 105 allogeneic irradiated PBMCs (lower chamber). Transwell plates (Nunclon, Rochester, NY) were gently placed in the lower chamber and 2 × 104 CD4+ CD25+ CD127 T cells with or without pre-incubation with RBV were plated in transwell plates with 1 × 105 allogeneic irradiated PBMCs (upper chamber). Soluble OKT3 20 μg/ml was added to both chambers and incubated for 7 days at 37°. At the end of incubation, the upper chamber was removed gently, and then 1 μCi of thymidine was added to the lower chamber and incubated for an additional 16 hr. Cells were harvested and [3H]thymidine incorporation was measured.

Statistics

Student's t-test and Bonferroni's multiple-comparison test were performed to analyse the significance of differences between groups in this study using graphpad prism (GraphPad Software, La Jolla, CA). All experiments were repeated five times, and a P value of < 0·05 was considered to represent a statistically significant difference.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

RBV down-modulated inhibitory activity of CD4+ CD25+ CD127 Treg cells against CD4+ CD25 Th cells

Before subsequent analysis, we confirmed the expression of FOXP3 in the isolated CD4+ CD25+ CD127 T cells and found that about 95% of them expressed FOXP3. No FOXP3 expression was seen in CD4+ CD25 T cells (Fig. 1a). The proliferation of CD4+ CD25 T cells was markedly inhibited when they were incubated for 7 days in the presence of CD4+ CD25+ CD127 T cells (Fig. 1b), confirming that the isolated CD4+ CD25+ CD127 T cells were phenotypically and functionally Treg cells.

image

Figure 1. Characteristics of CD25 or CD25+ CD127 CD4+ T cells used in this study. Peripheral blood mononuclear cells (PBMCs) were incubated with magnetic bead-conjugated antibodies without anti-CD4 monoclonal antibodies (mAbs), and the pass-through fraction was collected as CD4+ T cells. Subsequently, the CD4+ T-cell-containing fraction was incubated with FITC-conjugated anti-human CD25 together with Alexa-Fluor647-conjugated anti-human CD127 mAbs, and the CD25 and CD25+ CD127 fractions were obtained using FACSort. (a) Flow cytometric analysis was performed to confirm cell surface molecule expression on the isolated T cells. The expression of cell surface CD25, CD127 and intracellular FOXP3 is shown. The results for CD4+ CD25 T cells are shown in the upper two panels, and those for CD4+ CD25+ CD127 T cells are shown in the lower two panels. The purity of CD25+ CD127 T cells used in this study was approximately 95% (lower right panel). (b) The inhibitory activity of CD4+ CD25+ CD127 T cells against the proliferation of CD4+ CD25 T cells was confirmed; 2 × 104 CD4+ CD25 (empty column) and CD4+ CD25+ CD127 (filled column) T cells were re-stimulated in 96-well U-bottomed culture plates with pB human anti-CD3 mAb 0·05 μg/ml in the presence of 2 × 105 allogeneic irradiated PBMCs. Cell proliferation of CD4+ CD25 and CD4+ CD25+ CD127 T cells was determined 3, 5 and 7 days after stimulation in [3H]thymidine incorporation assays. The cell proliferation of the mixed culture of both cells (shaded column) was also measured. Bonferroni's multiple-comparison test was used to determine the significance of differences between groups.

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Next, we examined whether RBV affected the characteristics and regulatory activity of CD4+ CD25+ CD127 T cells. The cell viability of CD4+ CD25 and CD4+ CD25+ CD127 T cells was decreased when they were treated with RBV without stimulation. The numbers of viable CD4+ CD25+ CD127 T cells decreased more than that of CD4+ CD25 T cells (Fig. 2a). For this reason, we counted only the viable cells for use in the subsequent experiments. Intracellular FOXP3 expression in CD4+ CD25+ CD127 T cells was decreased when they were treated with RBV without stimulation (Fig. 2b, upper panels). In addition, the cell surface expression of ICOS was also decreased (Fig. 2b, lower panel). In contrast, CD28 expressed constitutively on the cell surface did not change after RBV incubation (data not shown). Although the proliferation of CD4+ CD25 and CD4+ CD25+ CD127 T cells did not change when they were incubated with RBV (Fig. 2c, left), the proliferation of CD4+ CD25 T cells, which was reduced in the presence of CD4+ CD25+ CD127 T cells, was clearly restored when they were incubated with CD4+ CD25+ CD127 T cells pre-incubated with RBV in an RBV dose-dependent manner when they were stimulated with a sub-optimal dose of human OKT3 (Fig. 2c, centre). A similar result was seen when the cells were stimulated with the maximum dose (5·0 μg/ml) of OKT3 (Fig. 2c, right).

image

Figure 2. Effects of ribavirin (RBV) on the characteristics of CD4+ CD25+ CD127 T cells. (a) The effect of RBV against cell viability of the isolated cells was confirmed. Each of the isolated cells was incubated with RBV 0 or 500 ng/ml for 48 hr without stimulation. The dead cells were confirmed as PI-stained cells in flow cytometry and their percentage is indicated. (b) The effects of RBV against the phenotype of the isolated cells were analysed. The isolated CD4+ CD25 and CD4+ CD25+ CD127 T cells were incubated with RBV 0 or 500 ng/ml for 48 hr. The change in FOXP3 expression in CD4+ CD25+ CD127 T cells is indicated in the upper two panels. The expression of inducible co-stimulatory molecule (ICOS) on the surface of the isolated cells is indicated in the lower two panels. ICOS expression of the unstained control (hatched line), on the cells without RBV (bold line), and that with RBV (shaded line) is indicated. (c) The effects of RBV alone against the proliferation of CD4+ CD25 and CD4+ CD25+ CD127 T cells were analysed. Each type of cells was pre-incubated with RBV 0–500 ng/ml for 48 hr and stimulated with 0·05 μg/ml human anti-CD3 mAbs in the presence of irradiated allogeneic PBMCs for 7 days and cell proliferation was confirmed. Student's t-test was used to determine the significance of differences between groups (c, left panel). The inhibitory activity of CD4+ CD25+ CD127 T-cells preincubated with RBV 0–500 ng/ml was evaluated. 104 CD4+ CD25 and CD4+ CD25+ CD127 T-cells preincubated with RBV 0–500 ng/ml were mixed and stimulated with a low dose (0·05 μg/ml, c, centre panel) and maximal dose (5·0 μg/ml, c, right panel) of human anti-CD3 monoclonal antibody in the presence of irradiated allogeneic peripheral blood mononuclear cells for 7 days, and the proliferation of these mixed cells was determined. Bonferroni's multiple-comparison test was used to determine the significance of differences between groups. Each experiment was performed five times, and the results are shown as mean and SD.

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RBV interfered with the conversion of CD4+ CD25 FOXP3 naive T cells into CD4+ CD25+ FOXP3+ Tregadapt cells

Intracellular FOXP3, a specific marker of Treg cells, can be induced in naive CD4+ T cells when stimulated with Tregnat cells. Moreover, our results showed that FOXP3 expression was slightly decreased in CD4+ CD25+ CD127 T cells when they were incubated with RBV. It was therefore expected that Treg cells pre-incubated with RBV could not induce the conversion of CD4+ CD25 FOXP3 T cells into CD4+ CD25+ FOXP3+ T cells. To confirm this hypothesis, we compared FOXP3 expression in CD4+ CD25 T cells stimulated with either CD4+ CD25+ CD127 T cells or those pre-incubated with RBV. FOXP3 was rarely expressed in CD4+ CD25 T cells when they were stimulated alone (Fig. 3a, upper left), and RBV had little effect on the expression of FOXP3 in either CD4+ CD25 (Fig. 3a, upper right) or CD4+ CD25+ CD127 T cells (Fig. 3a, centre right and left) after stimulation. CD25+ FOXP3+ cells increased when CD4+ CD25 T cells were stimulated with CD4+ CD25+ CD127 T cells (Fig. 3a, lower left). Surprisingly, these double-positive cells were markedly decreased when CD4+ CD25 T cells were stimulated with CD4+ CD25+ CD127 T cells pre-incubated with RBV (Fig. 3a, lower right). Mean numbers of CD25+ FOXP3+ cells were markedly reduced when CD4+ CD25 T cells were incubated with RBV-pre-incubated CD4+ CD25+ CD127 T cells, and the inhibition rate was 54·394 ± 11·975% (Fig. 3b). To confirm whether CD4+ CD25 T cells are activated or remain at rest in the presence of RBV, we also analysed the relationship between down-modulation of FOXP3 and the expression of the two CD45 isoforms CD45RA and CD45RO. Although the percentage of FOXP3+ CD45RO+ T cells was increased when CD4+ CD25 T cells were incubated with CD4+ CD25+ CD127 T cells, it was markedly decreased when CD4+ CD25 T cells were incubated with RBV-pre-incubated CD4+ CD25+ CD127 T cells without any decrease in the total counts of CD45RO+ cells (Fig. 3c).

image

Figure 3. Effects of ribavirin (RBV) on the ability of CD4+ CD25+ CD127 T cells to convert CD4+ CD25 T cells into CD4+ CD25+ FOXP3+ T cells. Intracellular FOXP3 was stained to confirm whether RBV modulated FOXP3 expression in CD4+ CD25 T cells after stimulation for 7 days with CD4+ CD25+ CD127 T cells with or without RBV pre-incubation. Cell surface CD25 and intracellular FOXP3 were stained on CD4+ CD25 T cells (a, upper-left panel), CD4+ CD25 T cells pre-incubated with RBV 500 ng/ml (upper right), CD4+ CD25+ CD127 T cells (centre left), CD4+ CD25+ CD127 T cells pre-incubated with RBV (centre right), CD4+ CD25 and CD4+ CD25+ CD127 T cells (lower left), and CD4+ CD25 T cells and CD4+ CD25+ CD127 T cells pre-incubated with RBV (lower right). The experiments were repeated five times, and representative results are shown. (b) Mean counts of CD25+ FOXP3+ T cells were compared when CD25 T cells were incubated with CD25+ CD127 T cells or RBV-pulsed CD25+ CD127 T cells. The mean inhibition rate was 54·394 ± 11·975%. Bonferroni's multiple-comparison test was used to determine the significance of differences between groups. (c) Expressed CD45RA and CD45RO were stained together with intracellular FOXP3 to determine whether CD4+ CD25 T cells entered anergy in association with the expression of FOXP3 or did not respond to re-stimulation in vitro. Left panels indicate the percentage of CD45RA+ and FOXP3+ cells in the indicated T cells. Right panels indicate the percentage of CD45RO+ and FOXP3+ cells.

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The inhibitory effect of RBV against CD4+ CD25+ FOXP3 T cells was transient

To confirm the inhibitory activity of CD4+ CD25 T cells incubated with CD4+ CD25+ CD127 T cells pre-incubated with 0 or 500 ng/ml of RBV, whole cells including CD4+ CD25 and CD4+ CD25+ CD127 T cells or those pre-incubated with RBV after a 7-day stimulation were mixed with freshly isolated CD4+ CD25 T cells and re-stimulated for 7 days with 0·05 μg/μl of anti-human CD3 mAb in the presence of irradiated allogeneic PBMCs. The cell viability rate of the collected cells after a 7-day incubation were 80–90%. Percentages of CD25+ CD127 T cells in these two cultures were markedly low (Fig. 4a, two left panels) and those of CD25+ FOXP3+ T cells did not change when CD25+ CD127 T cells were pre-treated with RBV (Fig. 4a, two right panels). The thymidine incorporation assay indicated that CD4+ CD25 T cells incubated with RBV-pulsed or unpulsed CD4+ CD25+ CD127 T cells inhibited the freshly isolated CD4+ CD25 T cells (Fig. 4b).

image

Figure 4. Changes in the inhibitory activity of the converted CD4+ T cells against freshly-isolated CD4+ CD25 T cells. The inhibitory effect of the primary-stimulated CD4+ CD25 and CD4+ CD25+ CD127 T cells was examined. The mixed cultures of CD4+ CD25 and CD4+ CD25+ CD127 T cells pre-incubated with or without ribavirin (RBV) 500 ng/ml were harvested, mixed with freshly isolated CD4+ CD25 T cells, and additionally incubated with 0·05 μg/ml of human anti-CD3 monoclonal antibodies in the presence of irradiated allogeneic peripheral blood mononuclear cells for 7 days. (a) Expression of CD25. CD127 and intracellular FOXP3 was confirmed. (b) Thymidine incorporation of the cultured cells including the indicated cells was measured. Bonferroni's multiple-comparison test was used to determine the significance of differences between groups.

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RBV inhibited activities of Treg cells by interfering with humoral elements released from Treg cells in a cell contact-dependent fashion

Because human Treg cells exhibit inhibitory activity in a contact-dependent and contact-independent fashion, it was important to determine whether RBV inhibited either or both of these cell types. The transwell system was used to separate CD4+ CD25 and CD4+ CD25+ CD127 T cells, and the thymidine incorporation assays for CD4+ CD25 T cells in the lower chambers showed that CD4+ CD25+ CD127 T cells also exhibited regulatory activity against CD4+ CD25 T cells even when they were separated, but the level of inhibitory activity was markedly diminished. Moreover, CD4+ CD25+ CD127 T cells pre-incubated with RBV did not inhibit the proliferation of CD4+ CD25 T cells in either mixed or separated culture conditions (Fig. 5).

image

Figure 5. Ribavirin (RBV) inhibited activities of CD4+ CD25+ CD127 T cells by interfering with humoral elements released from CD4+ CD25+ CD127 T cells in a cell contact-dependent fashion. Transwell assays were performed to determine whether direct cell contact was required for the inhibitory activity of CD4+ CD25+ CD127 T cells with or without RBV pre-incubation. CD4+ CD25+ CD127 T cells with or without RBV pre-incubation were plated in the upper chamber, and CD4+ CD25 T cells were plated in the lower chamber. Cells were stimulated with soluble anti-CD3 monoclonal antibody 20 μg/ml in the presence of irradiated allogeneic peripheral blood mononuclear cells. After 7 days of stimulation, the upper chamber was removed, and cell proliferation of the CD4+ CD25 T cells in the lower chamber was determined. Thymidine incorporation assays were performed to determine proliferation of the indicated cell fractions. Bonferroni's multiple-comparison test was used to determine the significance of differences between groups.

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RBV inhibited the activity of Treg cells through modulation of IL-10

To determine the key cytokine for the regulatory effects of CD4+ CD25+ CD127 T cells, we measured the levels of IL-10 and TGF-β1, the principal cytokines through which human Tregadapt cells exert regulatory activity, released from these cells after stimulation in vitro. The levels of IL-10 released from CD4+ CD25+ CD127 T cells were decreased when they were stimulated in the presence of RBV (Fig. 6a, upper panel). In contrast, the production of TGF-β1 was not decreased significantly (Fig. 6a, lower panel). We also examined the impact of these cytokines on CD4+ CD25+ CD127 T cells using their neutralizing mAbs. The reduced proliferation of CD4+ CD25 T cells in the presence of CD4+ CD25+ CD127 T cells was restored when they were incubated with anti-IL-10 mAbs. In addition, the restored proliferation of CD4+ CD25 T cells when stimulated with CD4+ CD25+ CD127 T cells pre-incubated with RBV was markedly decreased when they were stimulated in the presence of recombinant IL-10. In contrast, no effect was seen when the cells were stimulated in the presence of anti-TGF-β1 mAbs (Fig. 6b).

image

Figure 6. Inhibitory effects of ribavirin (RBV) against the production of inhibitory cytokines released from CD4+ CD25 or CD4+ CD25+ CD127 T cells. (a) To confirm the key cytokine that RBV affects to inhibit the activity of CD25+ CD127 T cells, levels of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) produced by CD4+ CD25 or CD4+ CD25+ CD127 T cells stimulated with human anti-CD3 monoclonal antibodies (mAbs) 5 ng/ml in the presence or absence of RBV were measured using ELISA. Student's t-test was used to determine the significance of differences between groups. (b) Neutralization of either IL-10 or TGF-β1 using their neutralizing antibodies was performed to determine which was the key cytokine for the inhibitory activity of CD4+ CD25+ CD127 T cells. CD4+ CD25 T cells were stimulated with the same number of CD4+ CD25+ CD12 T cells with pB anti-CD3 mAb 0·05 μg/ml in the presence or absence of either anti-IL-10 or anti-TGF-β1 mAbs for 7 days. Cell proliferation indicated below the graph was then determined in thymidine incorporation assays. Bonferroni's multiple-comparison test was used to determine the significance of differences between groups.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

In this study, we found that RBV down-modulated the inhibitory activity of human CD4+ CD25+ CD127 T cells (Treg cells) and also found that RBV interfered with the differentiation of CD4+ CD25 FOXP3 naive Th cells into CD4+ CD25+ FOXP3+ Tregadapt cells. Although the conversion of naive Th cells into Tregadapt cells is considered advantageous in terminating excessive activation of the cellular immune response against foreign antigens, it is disadvantageous in eliminating persistent pathogen infection because the increase in Treg cells down-modulates the pathogen-specific cellular immune response mediated by Th1 cells. Hence, the activity of RBV is considered appropriate for the elimination of persistent viral infections such as HCV, because blocking the differentiation of naive Th cells into Tregadapt cells allows the maintenance of Th1 cell activity without entering anergy, which may enhance the ability of HCV-specific CD8+ T cells to abrogate HCV-infected hepatocytes.

Our results indicated that Treg cells pre-incubated with RBV did not exhibit inhibitory activity against Th cells. Although it is still debatable which naive Th cells cannot differentiate or become unresponsive in the presence of Treg cells pre-incubated with RBV, the expression of CD45RO, known to be expressed on the surface of mature T cells,[34, 35] was unchanged when Th cells were incubated with Treg cells with or without pre-incubation with RBV, suggesting that naive T cells had been already stimulated. In addition, intracellular FOXP3 expression was significantly decreased in these cells when Th cells were incubated with Treg cells pre-incubated with RBV, suggesting that naive T cells had been stimulated but could not convert into Tregadapt cells when they were incubated with Treg cells pre-incubated with RBV.

The transwell assay system indicated that the inhibitory activity of Treg cells was diminished when their contact with Th cells was disrupted. Moreover, the proliferative activity of Th cells placed in the lower chamber of the transwell system was also restored when Treg cells placed in the upper chamber were pre-incubated with RBV. These results suggest that the inhibitory activity of Treg cells in this study depended on both cell contact and humoral elements released from Treg cells, and RBV seemed to inhibit both types of Treg cells. The Treg cells are known to comprise various subsets.[36-38] The Th3 cells, characterized phenotypically by their expression of glucocorticoid-induced tumour necrosis factor receptor (GITR) and absence of FOXP3, exhibit inhibitory activity in a TGF-β1-dependent manner and play an important role in inducing oral tolerance.[36] The other subset of Treg1 cells expresses FOXP3, produces both TGF-β1 and IL-10, and is activated in an IL-10-dependent manner.[37, 38] The CD4+ CD25+ CD127 T cells isolated in this study expressed high levels of FOXP3. Although they produced both IL-10 and TGF-β1, their inhibitory activity was significantly reduced only when they were incubated with anti-IL-10 mAbs. Moreover, RBV almost completely inhibited IL-10 release from CD4+ CD25+ CD127 T cells without affecting the release of TGF-β1. These results suggest that the CD4+ CD25+ CD127 T cells that we isolated exhibited both Tregnat and Treg1-cell-like characteristics. Because the main population of Treg cells in human peripheral blood is reported to comprise Tregnat cells, we tried to confirm that the intracellular FOXP3 and IL-10 double-positive cells in peripheral CD4+ CD25+ CD127 T cells were Treg1 cells. However, this was not possible because the expression of intracellular IL-10 was very low. It is difficult to isolate Treg1 cells phenotypically because both Tregnat cells and Treg1 cells express both CD25 and FOXP3.[39] In addition, because the main source of TGF-β1 in CD4+ T cells is known to be Treg cells, our result showing that CD4+ CD25 T cells released the same amount of TGF-β1 as CD4+ CD25+ CD127 T cells was confusing. However, some reports indicated that both Th cells and Treg cells released the same amount of TGF-β1.[40] Hence, further analysis will be needed to resolve this problem.

It remains uncertain how RBV inhibits Treg cells. Previous reports showed that RBV inhibits RNA synthesis by reducing nucleotide pooling in the host cells.[41, 42] Our results showing that RBV down-modulated cell surface ICOS and intracellular FOXP3 expression, which were inducible after cell stimulation, without affecting constitutively expressed molecules such as CD28 suggested that RBV may inhibit RNA synthesis, leading to down-modulation of FOXP3 expression, which is closely associated with Treg cell activity.[43] This may reduce the inhibitory activity of Tregnat cells along with down-modulating IL-10 secretion in Treg1 cells, which would in turn interfere with the differentiation of naive Th cells into Tregadapt cells. In addition, the effect of RBV on Treg cells appears to be transient because the inhibitory effect of Treg cells pre-treated with RBV was restored in association with the recovery of CD4+ CD25+ CD127 and intracellular FOXP3+ T cells. These results suggest that maintenance of the RBV concentration is required for continuous Treg cell inhibition. Because these results did not fully confirm the mechanism of action of RBV against immune regulatory cells, further analysis to determine the effects of RBV against other regulatory T cells will be required.

The RBV also inhibited the amount of IL-10 released from CD4+ CD25 T cells, suggesting that RBV has some effect on the characteristics of Th cells and other lymphocytes. We previously showed that RBV down-modulated ICOS expression on CD4+ Th cells, which was associated with a decrease in IL-10 released by them, leading to inhibition of differentiation of naive Th0 cells to Th2 cells.[30] The effect of RBV against the immune regulatory system therefore appears to be complicated. We could not confirm the details completely because we focused on the impact of RBV against Treg cells in this study. However, RBV could not modulate FOXP3 expression in Th cells, suggesting that the interference with the conversion of Th cells into Tregadapt cells is mainly associated with the RBV-induced down-modulation of Treg cells.

About 80% of HCV-infected patients have persistent HCV infection, which is the major cause of progressive liver injury leading to the development of cirrhosis.[44] Similar to other viruses, the eradication of HCV requires a complicated interaction between innate and acquired immune responses,[45] and various immune impairments are known to make HCV elimination difficult. Among them, the inappropriate activation of CD4+ and CD8+ T cells,[46] together with the impaired responses of dendritic cells against HCV,[47, 48] are associated with persistent HCV infection. The characteristics of Treg cells are also involved in persistent HCV infection. An increase in Treg cell number during acute HCV infection was reported to be closely associated with the failure to eradicate HCV.[49, 50] An increased frequency of FOXP3+ Treg cells was found in patients with chronic HCV infection.[51] Another report indicated the participation of both Treg1 and Th3 cells in persistent HCV infection.[24] In addition, the results of animal experiments suggested that HCV infection induces the differentiation of CD4+ CD25 T cells into CD4+ CD25+ Treg cells.[52] Based on those previous results, the activation of Treg cells against HCV antigen and the subsequent promotion of the differentiation of peripheral CD4+ CD25 T cells into CD4+ CD25+ FOXP3+ Tregadapt cells are considered to be critical for HCV infection to persist. Our results showing that RBV prevents the conversion of naive Th cells into Tregadapt cells indicate that RBV maintains Th1 cells in the activated phase, which enhances the eradication of HCV-infected hepatocytes. This is one potential mechanism by which RBV enhances HCV elimination in combination with IFN administration.

It was reported that Treg cells can be modulated by other drugs. The administration of low-dose cyclophosphamide (CPA), a chemotherapeutic reagent, enhanced cellular immune responses in mice[53] by its effects on Treg cells via induction of their apoptosis and down-modulation of both GITR and Foxp3 expression. Other reports indicated that Tregadapt cells expressed high levels of cyclooxygenase-2 (COX2) and could be enhanced in a prostaglandin-E2-dependent manner.[54, 55] Hence, COX2 inhibitors may be potential inhibitors of CD4+ CD25+ FOXP3+ Tregadapt cells.[55] Our results confirmed that RBV is a new reagent that down-modulates Treg cells through conversion of naive Th cells into Treg cells. This inhibitory activity against Treg cells was similar to that of CPA. These two reagents selectively down-modulate Treg cells without any effect on other effector lymphocytes. However, we did not investigate whether RBV induces apoptosis in Treg cells and did not clarify in detail how RBV modulates Treg cells, and therefore could not determine whether CPA or RBV was more effective in modulating Treg cell activity.

The ability of RBV to modulate Treg cells could be applied to the treatment of other diseases associated with immunological impairment. It was reported that there is a relationship between the down-modulation of Treg cells and the disease activity of systemic lupus erythematosus.[56] The ability of RBV to inhibit Treg cells would accelerate the activation of self-reactive Th cells in patients with systemic lupus erythematosus. Autoimmune liver disease, such as autoimmune hepatitis or primary biliary cirrhosis, is also associated with excessive activation of self-reactive T cells induced by the hypo-activity of Treg cells.[57, 58] Our results suggest that the administration of RBV in combination with IFN for the treatment of patients with HCV infection complicated by autoimmune hepatitis or primary biliary cirrhosis would accelerate self-reactive T-cell activation in association with down-modulation of Treg cells. In contrast, because tumour-associated antigen (TAA) is considered to be a self-generated antigen,[59] the TAA-specific cellular immune response would be suppressed if Treg cells corresponding to TAA-specific Th cells were activated to cause the Th cells to enter anergy.[60, 61] This is one essential mechanism that prevents the tumour-specific immune response in tumour-bearing hosts, and the reason why the regulation of Treg-cell activity will be useful for inducing tumour-specific immune responses.[62-65] Our results suggest that RBV enhances the TAA-specific cellular immune response in association with down-modulation of Treg-cell activity. As previously reported for CPA,[66] this hypothesis may contribute to preventing the progression to hepatocellular carcinoma in patients with HCV infection who were successfully treated with IFN plus RBV. To confirm this hypothesis, long-term observation of patients receiving pegylated IFN plus RBV therapy will be needed. In addition, it must be determined whether continuous administration of RBV after the elimination of HCV can contribute to the prevention of hepatocellular carcinoma.

In this report, we demonstrated the ability of RBV to inhibit the differentiation of naive CD4+ T cells into CD25+ FOXP3+ Tregadapt cells through the inhibition of Treg1-type regulatory cells. Although the mechanism of action by which RBV regulates Treg cells is not fully understood, we expect that these findings will contribute to establishing a new approach for regulating immune responses in patients with various diseases caused by immunological impairment.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

We are grateful to Dr Taku Tsukui, Division of Gastroenterology, Department of Medicine, Nippon Medical School, Tokyo, Japan, for critical reading of this manuscript and helpful suggestions.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References