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Keywords:

  • CD4+ CD25+ T cells;
  • FoxP3;
  • immune tolerance;
  • TGF-β2-treated APC

Summary

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

A growing body of evidence has shown that professional antigen-presenting cells (APC) treated with transforming growth factor-β (TGF-β) can induce a systemic antigen (Ag)-specific tolerance, similar to anterior chamber-associated immune deviation (ACAID). However, the exact mechanism for immune tolerance induced by TGF-β-treated APC has not been elucidated. In this study, we showed that intravenous injection of ovalbumin (OVA)-pulsed APC treated with TGF-β2 induced a peripheral tolerance as evidenced by an impaired delayed-type hypersensitivity (DTH) response. CD4+ T cells from mice receiving an intravenous injection of TGF-β2-treated APC pulsed with OVA could adoptively transfer a specific tolerance to naïve mice. An increased frequency of FoxP3-expressing CD4+ CD25+ T cells was observed in mice with tolerance. CD4CD25+ T cells from TGF-β2-treated APC-injected mice produced a large amount of TGF-β1 and exhibited an in vitro antigen-specific suppressive activity. CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice were able to inhibit the antigen-specific DTH response significantly when adoptively transferred to naïve mice. These results indicate that FoxP3-expressing CD4+ CD25+ T cells might be actively involved in the development of tolerance induced by TGF-β2-treated APC.


Introduction

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

It is generally accepted that professional antigen-presenting cells (APC) exposed to certain cytokines may be converted into a tolerogenic phenotype. Streilein and colleagues1–3 have suggested that intraocular F4/80+ APC, when exposed to the immunosuppressive microenvironment in the normal aqueous humour, become tolerogenic. These antigen-carrying APC migrate to the draining lymph nodes and spleen where they induce regulatory T cells, thereby mediating systemic antigen-specific tolerance [anterior chamber-associated immune deviation (ACAID)]. APC obtained from other sites, such as the peritoneal cavity, can also be converted into tolerance-inducing cells. Adoptive transfer of these APC treated with transforming growth factor-β2 (TGF-β2) and pulsed with antigen (Ag) can induce a systemic Ag-specific tolerance similar to ACAID.3–8 Although the APCs responsible for tolerance induction have been extensively studied during recent years, the exact mechanisms involved in the TGF-β2-treated APC-induced tolerance, as well as in ACAID, are still not fully understood.

The development of tolerance in vivo has been described as a highly dynamic process involving several mechanisms, including deletion of the alloreactive T-cell pool, T-cell anergy and immunoregulation.9 As a key component in peripheral tolerance, regulatory T cells are believed to be critical in the prevention of autoimmune diseases, allergies, transplant rejection and immune disorders.10,11 Recent studies indicate that multiple types of regulatory T cells are involved in the regulation of tolerance and that different subsets of CD4+ and CD8+ T cells participate in these regulatory activities.11–13 Of all types of regulatory cells, CD4+ CD25+ regulatory T cells are well studied and have been shown to be capable of suppressing a wide variety of immune cells in both the innate and adaptive immune systems.14–16 These cells are generally considered to be responsible for the immunoregulation of different forms of tolerance, such as oral tolerance and intravenous tolerance.17 However, whether CD4+ CD25+ regulatory cells are involved in the development of the tolerance induced by TGF-β2-treated APC remains unknown.

Several studies have demonstrated the crucial role of a transcription factor, FoxP3, in the development of CD4+ CD25+ regulatory T cells.18–20 As indicated by recent in vivo and in vitro studies, the expression of FoxP3, also known as scurfin, is mainly restricted to CD4+ CD25+ regulatory T cells. Deficiency in the function of FoxP3 protein results in a specific loss of CD4+ CD25+ regulatory T cells and leads to the development of a variety of autoimmune disorders. In addition, naive CD4+ T cells, when forced to express FoxP3, were found to acquire suppressor properties.21–23

Recent studies have shown that TGF-β, a major component of the immunosuppressive environment in the normal aqueous humour, plays an important role in inducing the generation of CD4+ CD25+ regulatory T cells. Treatment of peripheral CD4+ CD25+ T cells with TGF-β2 has been shown to induce the expression of FoxP3 and the upregulation of CD25.24–26 Exposing naive DO11.10 T cells in vitro to active TGF-β can induce the generation of CD4+ CD25+ regulatory T cells.27 In the present study, we investigated whether CD4+ CD25+ regulatory T cells are associated with the development of tolerance induced by TGF-β2-treated APC. We showed that the proportion of FoxP3 CD25+ within CD4+ T cells was up-regulated in the spleen of mice in which tolerance had been induced by the intravenous (i.v.) injection of TGF-β2-treated APC. The CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice exhibited an Ag-specific suppressive role in cell proliferation and a delayed-type hypersensitivity (DTH) response in association with an increased secretion of TGF-β1.

Materials and methods

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

Animals

Female BALB/c mice (6–8 weeks old) were purchased from the animal center of the Sun Yat-sen University (Guangzhou, China). Mice were housed and cared for in a pathogen-free facility according to National Institute of Health Guidelines and the ARVO Statement for the Use of Animals in Ophthalmology and Vision.

Isolation and preparation of tolerance-inducing macrophages (TGF-β-treated APC)

Peritoneal exudate cells were generated by an intraperitoneal injection of 3 ml of 3% thioglycolate (Sigma Chemical Co., St Louis, MO) 4 days before harvesting. The obtained cells were plated in six-well culture plates and cultured at 37° in an atmosphere of 5% CO2 for 2 hr. The non-adherent cells were removed and the adherent macrophages were treated with 5 mg/ml of ovalbumin (OVA; Sigma-Aldrich, St Louis, MO) and 2 ng/ml of human TGF-β2 (R&D Systems, Minneapolis, MN) in serum-free RPMI medium at 37° and 5% CO2 in air. Cells incubated with 5 mg/ml of OVA alone served as a control. After overnight culture, the plates were washed to remove TGF-β2 and non-adherent cells. Adherent cells were collected and used in the following experiments. Flow cytometric analysis using fluorescein isothiocyanate (FITC)-conjugated F4/80+ monoclonal antibody (mAb) (Serotec, Oxford, UK) was performed to identify the purity of the obtained adherent cells. The results showed that more than 90% of these cells were F4/80 positive.

Induction of tolerance

An i.v. injection of the aforementioned antigen-pulsed TGF-β2-treated APC (2 × 104 cells) was used to induce peripheral tolerance. Control mice were injected i.v. with either antigen-pulsed APC or phosphate-buffered saline (PBS). In addition, ACAID induced by an intracameral injection of antigen was used as experimental control. For the induction of ACAID, mice received a single intracameral injection of 100 μg of OVA in 5 μl of PBS, as described previously.1

Subcutaneous immunization and DTH assay

Seven days following i.v. injection of 2 × 104 TGF-β2-treated APC or anterior chamber inoculations of OVA, mice were challenged with a subcutaneous injection of 250 μg of OVA in PBS emulsified 1:1 in complete Freund’s adjuvant (CFA; Sigma-Aldrich). Each mouse received a total volume of 200 μl.

For DTH assay, mice received an intradermal injection of 200 μg of OVA in 20 μl of PBS in the right ear pinnae 7 days after immunization. The left ear pinnae received 20 μl of sterile PBS alone. Both ear pinnae were measured using a Mitutoyo engineer’s micrometer (Mitutoyo, Aurora, IL) before and 24 hr after the challenge. The difference in thickness was used as a measure of the DTH response. Results were expressed as: specific ear swelling = [(24-hr measurement − 0-hr measurement) of the right ear − (24-hr measurement − 0-hr measurement) of the left ear]. Mice receiving an ear challenge only were used as negative controls. Mice receiving a subcutaneous injection of OVA and CFA served as positive controls.

Analysis of frequencies of CD4+ CD25+ and FoxP3 by flow cytometry

Spleen cells from each group were prepared for phenotype analysis and intracellular staining. For analysis of CD4+ CD25+ subsets, the spleens from the experimental mice were removed and then pressed through a nylon mesh to produce a single-cell suspension. Red blood cells were lysed with lysis buffer (Tris–NH4Cl: 0·83% in 0·01 m Tris–HCl, pH 7·2). Cells were stained with phycoerythrin (PE)–cy7–anti-CD3, FITC–anti-CD4 and allophycocyanin (APC)–anti-CD25 (eBioscience, San Diego, CA) at 4° for 30 min, washed twice with PBS containing 0·5% bovine serum albumin (BSA) and fixed with 1% paraformaldehyde. The mean fluorescence intensity (MFI) value and positive cell percentages were determined by flow cytometry (facsdiva; Becton Dickinson Biosciences, San Jose, CA). For detection of intranuclear expression of FoxP3, cells were stained with PE–cy7–anti-CD3, FITC–anti-CD4 and APC–anti-CD25 for 30 min, fixed for 20 min at 4° in fixation buffer (eBioscience) and permeabilized in permeabilization buffer (eBioscience). The permeabilized cells were incubated with PE-labelled anti-FoxP3 (eBioscience) for 30 min at 4°. Negative controls were performed using PE-conjugated isotype antibodies (eBioscience). After washing once with the saponin buffer and twice with PBS, the labelled cells were detected by flow cytometry.

Enrichment of T-cell populations by magnetic affinity cell sorting (MACS)

CD4+ T cells were purified by magnetic separation (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. Briefly, splenocytes were prepared and non-CD4+ T cells were indirectly labelled with a cocktail of biotin-conjugated antibodies and anti-biotin MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). The cell suspension was loaded onto a MACS column, which was placed in the magnetic field of a MACS separator, and eluted from the column. The flow-through cells were shown by flow cytometric analysis to be enriched CD4+ T cells (92% positive).

For the enrichment of CD4+ CD25+ T cells, the splenocytes were labelled with a cocktail of biotin-conjugated antibodies followed by incubation with anti-biotin microbeads and PE-conjugated anti-mouse CD25 mAb at 4° for 15 min and then subjected to an LD column (Miltenyi Biotec, Auburn, CA). The flow-through cells (CD4+ cells) were then magnetically labelled with anti-PE microbeads and purified by double MS+ columns, as described by the manufacturer (Miltenyi Biotec, Auburn, CA). The retained cells were eluted from the column and shown, by flow cytometric analysis, to be enriched CD4+ CD25+ T cells (purity > 90%). The flow-through cells were shown to be the enriched CD4+ CD25 T cells (purity > 90%).

In vitro proliferation assay and cytokine assay

For the proliferation assay, mice were injected i.v. with 2 × 104 antigen-pulsed TGF-β2-treated APC and immunized with OVA/CFA 7 days later. Splenocytes were collected and CD4+ cells were purified from naïve mice, from positive-control mice and from mice receiving an i.v. injection of TGF-β2-treated APC 10 days after immunization. Purified CD4+ T cells (4 × 105 cells/well) from all the three groups were cultured with mitomycin C (MMC; Sigma-Aldrich)-treated spleen cells (8 × 105 cells/well, as APCs) in the absence or presence of varying doses of OVA in 96-well plates. In some experiments, purified CD4+ CD25 T cells (4 × 104 cells/well, as responder cells) from positive-control groups were co-cultured with different concentrations of purified CD4+ CD25+ T cells (as suppressor cells) from the naïve mice, positive-control mice and mice in whom tolerance had been induced by i.v. injection of TGF-β2-treated APC and MMC-treated spleen cells (2 × 105 cells/well, as APCs) in the absence or presence of 200 μg/ml of OVA. The ratio of CD4+ CD25 T cells to CD4+ CD25+ T cells was 1:0, 1:0·25, 1:0·5 and 1:1 respectively. In all T-cell proliferation assays, the plates were incubated at 37° for 72 hr and pulsed with 1 μCi of [3H]thymidine ([3H]TdR) (Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) for the last 16 hr before harvesting. All determinations were conducted in quadruplicate and [3H]TdR incorporation was determined.

For the detection of TGF-β1 and interleukin (IL)-10 production, freshly isolated splenic CD4+ T cells (1 × 106 cells/well), CD4+ CD25+ T cells (4 × 104 cells/well) and CD4+ CD25 T cells (4 × 104 cells/well) from naïve mice, positive-control mice and mice in whom tolerance had been induced by i.v. injection of TGF-β2-treated APC were cultured with MMC-treated spleen cells in round-bottom 96-well plates or in 24-well plates, and stimulated with or without 200 μg/ml of OVA for 72 hr. The supernatants were collected and stored at −80°. IL-10 in supernatants was measured using enzyme-linked immunosorbent assay (ELISA) kits (Bender, Vienna, Austria), and TGF-β1 was quantified using a human ELISA kit (R&D systems), following the manufacturer’s instructions. The sensitivities of the assays were 5·28 and 7 pg/ml respectively.

Adoptive transfer assay

TGF-β2-treated APC were injected into naive mice and the spleen cells were harvested 7 days later. CD4+ T cells, CD4+ CD25+ T cells and CD4+ CD25 T cells (2 × 106, 2 × 105 or 2 × 104) were purified and injected i.v. into naïve mice. The mice were challenged 3 days later, and the DTH response was determined according to the method described above.

Statistical analysis

Data were subjected to analysis by analysis of variance (anova) using spss 11.5 software (SPSS Science, Chicago, IL). A P-value of < 0·05 was considered significantly different. All experiments were performed in triplicate, and error bars represent the standard deviation (SD).

Results

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

Tolerance can be induced by intravenous injection of TGF-β2-treated APC

To induce tolerance, mice were injected i.v. with 2 × 104 TGF-β2-treated APC and tested by evaluating the DTH response following immunization with OVA and adjuvant on day 7. The DTH response was evaluated by measurement of ear swelling thickness. The results showed that mice receiving an i.v. injection of OVA-pulsed APC treated with TGF-β2 displayed an impaired DTH response. Furthermore, the results showed that OVA-pulsed TGF-β2-treated APC induced an impaired DTH response to OVA, but not to BSA. A similar result was also observed in mice receiving an intracameral injection of OVA. In contrast, mice receiving either an i.v. injection of OVA-pulsed APC or an i.v. injection of PBS showed a vigorous DTH response, which was similar to that in the positive controls (Fig. 1).

image

Figure 1.  Transforming growth factor-β2 (TGF-β2)-treated peritoneal exudate cells (PEC) inhibited the delayed-type hypersensitivity (DTH) response. Mice were injected intravenously (i.v.) with 2 × 104 ovalbumin (OVA)-pulsed antigen-presenting cells (APC) treated with TGF-β2, OVA-pulsed APC, or phosphate-buffered saline (PBS) alone, and then immunized with OVA/complete Freund’s adjuvant (CFA) on day 7. The mice were challenged by injection of OVA into the ear pinnae 7 days after immunization. Ear swelling was measured 24 hr later. In the anterior chamber-associated immune deviation (ACAID) groups, soluble OVA (100 μg) was injected intracamerally (i.c.) into naive mice. The mice were immunized subcutaneously (s.c) with OVA/CFA 7 days later. DTH was assessed as described in the text. Positive-control mice were immunized s.c. with OVA/ CFA. Data represent the mean ± standard deviation of three independent experiments with six mice in each group. **P < 0·01 for experimental groups versus positive controls.

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CD4 cells from mice receiving an i.v. injection of TGF-β2-treated APC can adoptively transfer tolerance

An adoptive transfer experiment was performed to test whether CD4+ cells from mice receiving an i.v. injection of TGF-β2-treated APC could induce immune tolerance. The results demonstrated that an impaired antigen-specific DTH response to OVA could be transferred by CD4+ cells from TGF-β2-treated APC-injected mice. On the other hand, CD4+ T cells from naïve mice did not induce tolerance when transferred to normal mice (Fig. 2).

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Figure 2.  Adoptive transfer of CD4+ T cells from transforming growth factor-β2 (TGF-β2)-treated peritoneal exudate cells (PEC)-injected mice to naïve mice induced immune tolerance. CD4+ cells were purified either from mice receiving injection of 2 × 104 antigen-pulsed TGF-β-treated antigen-presenting cells (APC) or naïve mice, and injected intravenously (i.v.) into naïve mice. The mice were immunized with ovalbumin (OVA)/complete Freund’s adjuvant (CFA) on day 3. The delayed-type hypersensitivity (DTH) response was assayed according to the method described in the text. Positive-control mice were immunized subcutaneously (s.c.) with OVA/ CFA. Data represent the mean ± standard deviation of three independent experiments with five mice in each group. *P < 0·05 or **P < 0·01 for experimental groups versus positive controls.

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CD4+ cells from mice receiving an i.v. injection of TGF-β2-treated APC possess antigen-specific suppressive ability in vitro

To characterize the CD4+ T-cell functions in vitro, we isolated CD4+ T cells from splenocytes by MACS and studied their proliferation activity and cytokine profiles. As shown in Fig. 3a, CD4+ T cells from TGF-β2-treated APC-injected mice showed a weaker proliferation than those from the positive control in response to stimulation with different doses of OVA in the presence of MMC-treated splenocytes as APCs (P < 0·05 at OVA doses of 100, 200 and 400 μg/ml). CD4+ T cells from naïve mice showed a very weak proliferation upon stimulation with OVA. No obvious proliferative response was observed in each group in the absence of OVA stimulation. Given the observation that no significant difference concerning proliferation was observed in response to stimulation with 200 μg/ml versus 400 μg/ml of OVA (P > 0·05), the 200 μg/ml dose of OVA was used for the subsequent cytokine experiments.

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Figure 3.  Transforming growth factor-β2 (TGF-β2)-treated peritoneal exudate cells (PEC) inhibit the proliferation of CD4+ T cells and induce the production of TGF-β1 and interleukin (IL)-10. Mice were injected intravenously (i.v.) with 2 × 104 antigen-pulsed TGF-β2-treated antigen-presenting cells (APC), then 7 days later were immunized with ovalbumin (OVA)/CFA. Ten days later, splenocytes were collected and CD4+ cells were purified from mice receiving i.v. injection of TGF-β2-treated PEC or OVA immunization or from naïve mice. CD4+ cells were cultured in the presence of mitomycin C (MMC)-treated APC and (a) various concentrations of OVA and the proliferation response were evaluated, or (b) cells were cultured in the presence of 200 μg/ml of OVA and the supernatant was collected 72 hr later and tested for TGF-β1 and IL-10 by enzyme-linked immunosorbent assay (ELISA). All data shown represent the mean ± standard deviation of three independent experiments. *P < 0·05 or **P < 0·01 for experimental groups versus naive groups. #P < 0·05 for experimental groups versus positive controls. c.p.m., counts per minute.

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CD4+ T cells from TGF-β2-treated APC-injected mice, when stimulated with or without OVA, produced higher levels of TGF-β1 and IL-10 compared with those from naïve and positive controls. The levels of TGF-β1 and IL-10 produced by CD4+ T cells from positive-control mice were also higher than those from naïve mice, upon stimulation with OVA. CD4+ T cells from neither naïve nor positive-control mice secreted detectable levels of TGF-β1 and IL-10 in the absence of OVA stimulation (Fig. 3b).

Intravenous injection of TGF-β2-treated APC results in an increased frequency of CD4+ CD25+ T cells

As CD4+ CD25+ T cells have been reported to possess a regulatory property, we analysed the level of these cells after i.v. injection of TGF-β2-treated APC using flow cytometry. As shown in Fig. 4a, there was an increased percentage of CD4+ CD25+ T cells within CD4+ T cells in the mice receiving an i.v. injection of TGF-β2-treated APC compared with that seen in naïve mice (P < 0·01). Similarly, an increased percentage of CD4+ CD25+ T cells was also observed in mice receiving immunization with OVA and adjuvant and in mice receiving an i.v. injection of TGF-β2-treated APC and immunization with OVA. Interestingly, the percentage increase in the latter group was higher than that observed in the former group (P < 0·05).

image

Figure 4.  Expression of CD25 and Foxp3 in CD4+ T cells of the spleen were analysed by flow cytometry (FCM). Spleen cells were prepared and labelled with phycoerythrin (PE)–cy7–anti-CD3, fluorescein isothiocyanate (FITC)–anti-CD4 and allophycocyanin (APC)–anti-CD25 and PE-labelled anti-FoxP3 monoclonal antibody (mAb). (a) The histogram shows the percentage of CD25+ on CD4+ T cells. (b) CD3CD4+ T cells were gated and analysed for CD25+ Foxp3+ expression. (c) The histogram shows the percentage of CD25+ Foxp3+ within CD4+ T cells. All data shown represent the mean ± standard deviation of three independent experiments with five mice in each group. **P < 0·01 for experimental groups versus naïve controls. #P < 0·05 for experimental groups versus positive controls.

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Intravenous injection of TGF-β2-treated APC promotes the expression of Foxp3 in CD4+ CD25+ T cells

As Foxp3 is a definitive marker for regulatory T cells, we tested the expression of Foxp3 in CD4+ CD25+ T cells during the tolerance induced by TGF-β2-treated APC. The results showed that Foxp3 was found to be expressed by more than 90% of CD4+ CD25+ T cells, but only by about 6% of CD4+ CD25 T cells. The proportions of CD25+ Foxp3+ T cells within CD4+ T cells in mice receiving an i.v. injection of TGF-β2-treated APC and in mice receiving an i.v. injection of TGF-β2-treated APC plus immunization were not different but were significantly higher than those in naïve mice (P < 0·01 and P < 0·01, respectively). The percentage of CD25+ Foxp3+ T cells within CD4+ T cells in the mice receiving an immunization with OVA and CFA (positive group) was similar to that observed in naïve mice (Fig. 4b,c). There were no differences concerning the MFI value of the Foxp3 protein on CD4+ CD25+ T cells among all three groups.

CD4+ CD25+ T cells from mice receiving an i.v. injection of TGF-β2-treated APC possess significant regulatory activity in vitro

As the increased percentage of CD4+ CD25+ Foxp3+ T cells in mice receiving an i.v. injection of TGF-β2-treated APC was demonstrated by the aforementioned studies and it was impossible to obtain these cells as regulatory cells in in vitro experiments, we further investigated the suppressive effect of purified CD4+ CD25+ T cells, rather than CD4+ CD25+ Foxp3+ T cells, from mice with tolerance, and from naïve and from positive-control mice, on the proliferation of CD4+ CD25 T cells. As shown in Fig. 5a, the CD4+ CD25+ T cells from mice receiving an i.v. injection of TGF-β2-treated APC, from naïve mice and from positive-control mice all showed an inhibitory effect on the proliferative response of CD4+ CD25 T cells. However, a stronger inhibitory activity was observed in CD4+ CD25+ T cells from mice receiving an i.v. injection of TGF-β2-treated APC compared with those from naïve or positive-control mice. Furthermore, this suppression was exhibited in a dose-dependent manner. The experiment without OVA stimulation revealed that CD4+ CD25 T cells from each group showed a weak proliferation and that CD4+ CD25+ T cells did not show any suppression on the proliferation of CD4+ CD25 T cells.

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Figure 5.  CD4+ CD25+ T cells from transforming growth factor-β2 (TGF-β2)-treated peritoneal exudate cells (PEC)-injected mice showed antigen (Ag)-specific suppression in vitro. (a) The suppressive effect of CD4+ CD25+ T cells on the proliferation of CD4+ CD25 T cells. Purified CD4+ CD25 T cells (2 × 104 cells/well, as responder cells) from positive control mice were co-cultured with purified CD4+ CD25+ T cells (as suppressor cells) from naïve mice, positive controls and TGF-β2-treated PEC-injected mice at different responder/suppressor ratios (1:0, 1:0·25, 1:0·5 or 1:1) in the presence of mitomycin C (MMC)-treated splenocytes from naïve mice [2 × 105 cells/well, as antigen-presenting cells (APCs)] and ovalbumin (OVA) (200 μg/ml) for 72 hr. [3H]Thymidine was added for the last 16 hr and incorporation of radioactivity was measured by scintillation counting. #P < 0·05 for TGF-β2-treated PEC-injected groups versus positive-control groups. (b) The production of TGF-β1 and IL-10 by CD4+ CD25+ T cells and CD4+ CD25 T cells. CD4+ CD25+ T cells and CD4+ CD25 T cells from naïve mice, positive-control mice and TGF-β2-treated PEC-injected mice were stimulated with or without OVA (200 μg/ml) in the presence of APCs for 72 hr respectively. TGF-β1 and interleukin (IL)-10 in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). All data shown represent the means ± standard deviation of three independent experiments. *P < 0·05 or **P < 0·01 for experimental groups versus naive groups. #P < 0·05 for experimental groups versus positive controls. c.p.m., counts per minute.

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As regulatory T cells may exert their effects through certain cytokines, we investigated the production of TGF-β and IL-10 by these CD4+ CD25+ T cells. The CD4+ CD25+ T cells from each group were co-cultured respectively with MMC-treated spleen cells in the presence or absence of OVA, and the levels of TGF-β1 and IL-10 in the supernatants of the cultures were subsequently measured by ELISA. The results showed that the CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice, when stimulated with OVA, produced a significantly higher level of TGF-β1 than those from naïve and positive-control mice (P < 0·01, P < 0·05 respectively). The level of TGF-β1 produced by CD4+ CD25+ T cells from positive-control mice was also higher than that from naïve mice. Furthermore, the T cells from TGF-β2-treated APC-injected mice also produced detectable amounts of TGF-β1, even without OVA stimulation. Neither cells from naïve mice nor those from positive-control mice secreted detectable TGF-β1 in the absence of OVA stimulation (Fig. 5b). No increased level of IL-10 produced by CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice was observed when stimulated with or without OVA (data not shown). On the other hand, we did find that CD4+ CD25 T cells from TGF-β2-treated APC-injected mice, when stimulated with or without OVA, produced a higher level of IL-10 than those from naïve mice or from positive-control mice (P < 0·01, P < 0·05 respectively). The level of IL-10 produced by CD4+ CD25 T cells from positive-control mice was also higher than those from naïve mice, upon stimulation with OVA. Neither cells from naïve mice nor those from positive-control mice secreted a detectable level of IL-10 without OVA stimulation (Fig. 5b).

CD4+ CD25+ T cells from mice receiving an i.v. injection of TGF-β2-treated APC could adoptively transfer tolerance

As CD4+ CD25+ T cells from mice receiving an i.v. injection of TGF-β2-treated APC were shown to have inhibitory properties in in vitro experiments, we further tested whether they also possess this function in vivo by using an adoptive-transfer experiment. As shown in Fig. 6, CD4+ CD25+ T cells markedly inhibited DTH responses to OVA, but not to BSA, when transferred to normal mice. A vigorous DTH response was detected when CD4+ CD25 T cells were transferred at a number equal to CD4+ CD25+ T cells. However, when a large number of CD4+ CD25 T cells (5 × 106 cells per mouse) from mice receiving an i.v. injection of TGF-β2-treated APC were transferred, DTH responses were also suppressed.

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Figure 6.  CD4+ CD25+ T cells from transforming growth factor-β2 (TGF-β2)-treated peritoneal exudate cells (PEC)-injected mice inhibit delayed-type hypersensitivity (DTH) responses in vivo. Splenic CD4+ CD25+ and CD4+ CD25 T cells from naïve mice, positive-control mice and TGF-β2-treated PEC-injected mice were purified by magnetic affinity cell sorting (MACS). Isolated CD4+ CD25+ T cells and CD4+ CD25 T cells were injected intravenously into the tail vein of syngeneic mice respectively. The recipients received a subcutaneous (s.c.) injection of ovalbumin (OVA) in complete Freund’s adjuvant (CFA) after 24 hr and were challenged by injection of OVA into the right ear pinnae on day 7. DTH was assessed as described in the text. Data represent the means ± standard deviation of three independent experiments. *< 0·05 or **P < 0·01 for experimental groups versus positive controls.

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Discussion

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

In the present study, we successfully induced an immune tolerance using i.v. injection of TGF-β2-treated APC, as evidenced by an impaired DTH response similar to that observed in ACAID. The frequencies of CD4+ CD25+ T cells and FoxP3-expressing CD4+ CD25+ T cells in splenocytes were increased in mice receiving an i.v. injection of TGF-β2-treated APC pulsed with OVA. These CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice showed an increased secretion of TGF-β1 and were more effective at suppressing the proliferation of CD4+ CD25 T cells than those from naïve mice and positive-control mice. Furthermore, these cells could adoptively transfer an antigen-specific tolerance to naïve mice. These results suggest that Foxp3-expressing CD4+ CD25+ regulatory T cells may be involved in the development of immune tolerance induced by TGF-β2-treated APC.

It is well known that professional APC can induce either an immune response or immune tolerance, principally depending on their stage of maturation or level of activation.28 Some cytokines, such as IL-10 and TGF-β, can modify the function of APC and convert them into a tolerogenic phenotype.1–3,29 Our results confirmed that peritoneal exudate cells, when treated with TGF-β2 and i.v. injected into mice, induced an impairment of DTH responses after immunization with OVA, whereas OVA-pulsed peritoneal exudate cells mediated a vigorous DTH response.

Ample evidence has shown that i.v. injection of TGF-β-treated APC induces a systemic and antigen-specific tolerance similar to that of ACAID.4–8 However, the mechanisms of immune tolerance induced by TGF-β-treated APC have not been fully clarified. A growing body of evidence suggests that CD4+ T cells are important regulators in different forms of immune tolerance. Recent studies have also shown that CD4+ T cells are involved in the generation of ACAID regulatory T cells.30,31 Consistent with these studies, our results showed that CD4+ T cells from mice receiving an i.v. injection of TGF-β2-treated APC could transfer tolerance to naive recipients, whereas CD4+ cells from naïve mice did not have this property. CD4+ T cells from TGF-β2-treated APC-injected mice showed a weak proliferation when simulated with varying doses of OVA and produced a larger amount of IL-10 than those from naïve mice and positive-control mice. Importantly, we found that CD4+ T cells from TGF-β2-treated APC-injected mice, when primed with OVA, also produced a higher level of TGF-β1, an important cytokine involved in the modulation of Foxp3 expression and regulatory activity in distinct CD4+ T-cell subsets.32,33 Together, these results suggest that regulatory subsets may be present in CD4+ T cells from TGF-β2-treated APC-injected mice and that they may mediate the immune tolerance observed in these mice. It is interesting to note that CD4+ T cells from positive-control mice were also able to produce low levels of TGF-β1 and IL-10. Studies in knockout mice have shown that several inhibitory factors, such as IL-10, TGF-β or cytotoxic T-lymphocyte antigen-4 (CTLA-4) could act as gatekeepers of the adaptive immune response. Lack of these inhibitory molecules leads to massive inflammatory responses mainly mediated by activated T cells.34,35 Therefore, the increased production of IL-10 and TGF-β1 by CD4+ T cells from the positive-control mice may reflect a downregulation of T-cell activity mediated by OVA and CFA immunization.

As CD4+ CD25+ T cells have been considered as the cells with an immunosuppressive property in CD4+ T cells, we further investigated the CD4+ CD25+ T cells phenotypically and functionally during tolerance induced by TGF-β2-treated APC. Our results showed that the frequency of CD4+ CD25+ T cells in the spleen was increased during the development of tolerance induced by the i.v. injection of TGF-β2-treated APC. Foxp3, a crucial regulatory gene for the development and function of regulatory T cells, was found to be specifically expressed in CD4+ CD25+ T cells. Increased percentages of Foxp3-expressing CD25+ T cells within CD4+ T cells were observed in the mice receiving i.v. injection of TGF-β2-treated APC, but not in naive mice and positive-control mice. Immunization with OVA and CFA did not influence the expression of Foxp3 in CD4+ CD25+ T cells, consistent with previous reports that the Foxp3 gene could not be upregulated in recently activated CD4+ CD25 T cells. Furthermore, flow cytometric analysis showed that there were no significant differences concerning the MFI value for Foxp3 expression in CD4+ CD25+ T cells among the different groups. These results indicate that i.v. injection of TGF-β2-treated APC causes an increase in the number of Foxp3-expressing CD25+ T cells within CD4+ T cells, but does not up-regulate the expression of Foxp3 protein in CD25+ T cells. The increased percentage of Foxp3-expressing CD25+ T cells may be induced by the treatment with TGF-β2-treated APC.

Functional studies showed that CD4+ CD25+ T cells from receiving i.v. injection of TGF-β2-treated APC were hypoproliferative, inhibited the proliferation of CD4+ CD25 T cells upon stimulation with the insulting Ag and could induce an Ag-specific inhibition of the DTH response, when adoptively transferred into naïve mice. These CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice were found to produce large amounts of TGF-β1. Recent studies have demonstrated that TGF-β1 signaling is required for the maintenance of Foxp3 expression and suppressive activity of CD4+ CD25+ regulatory T cells.36–38 It is more likely that TGF-β-producing CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice exert regulatory activities, thereby contributing to the induction of tolerance.

There is substantial evidence that natural CD4+ CD25+ regulatory T cells arise in the thymus and contribute to the maintenance of immunological tolerance. Functional CD4+ CD25+ regulatory T cells can also be induced by TGF-β, immature dendritic cells, or IL-10. Several in vitro studies have shown that TGF-β could help the conversion of in vitro-stimulated peripheral CD4+ CD25 T cells into Foxp3-expressing suppressor cells. Naturally occurring CD4+ CD25+ regulatory T cells can directly induce conventional CD4+ cells to become suppressive cells following contact with IL-2 and TGF-β.39–45 Therefore, it is possible that abundant TGF-β1 produced by CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice might be involved in the antigen-driven conversion of suppressor cells in vivo. However, this presumption remains to be further clarified. It is worth pointing out that CD4+ CD25+ T cells from positive-control mice and naïve mice also showed a weak inhibitory effect on the proliferation of CD4+ CD25 T cells. This result indicates the functional involvement of natural CD4+ CD25+ regulatory T cells within the CD4+ CD25+ T-cell population from positive-control mice and naïve mice. The enhanced inhibitory capacity of CD4+ CD25+ T cells from positive-control mice might be associated with increased production of TGF-β1 as compared with those from naïve mice.

Unlike CD4+ CD25+ T cells, CD4+ CD25 T cells from TGF-β2-treated APC-injected mice did not show any suppressive activity in DTH responses when used at a number equal to CD4+ CD25+ T cells. Interestingly, a 10-fold higher number of CD4+ CD25 T cells from TGF-β2-treated APC-injected mice, when adoptively transferred to naïve mice, could also suppress DTH responses. Moreover, these cells were found to produce a higher level of IL-10 in mice receiving an i.v. injection of TGF-β2-treated APC compared with those from naïve mice and positive-control mice. Recent studies have suggested that a subset of IL-10-secreting regulatory cells is present in the CD4+ CD25 T cells and that they may also be involved in immune suppression.46–49 It seems likely that the IL-10-producing CD4+ T cells may also function as a subset of regulatory T cells and are involved in the development of the immune tolerance induced by i.v. injection of TGF-β2-treated APC. It is worth pointing out that this presumption should be clarified by blocking experiments with anti-IL-10 antibody. In addition, we observed that the relatively higher level of IL-10 was also produced by CD4+ CD25 T cells from positive-control mice. Whether the increased IL-10 production is involved in the downregulation of the T-cell reaction mediated by OVA and CFA immunization needs further investigation.

In conclusion, our study revealed that an immune tolerance could be induced in mice by i.v. injection with TGF-β2-treated APC. CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice represent an important subset of CD4+ regulatory T cells that are responsible for the development of tolerance induced by TGF-β2-treated APC, as evidenced by their suppressive role in cell proliferation, production of TGF-β1 and adoptive transfer of Ag-specific immune tolerance to naïve mice. Experiments with antibodies against TGF-β or IL-10 to block the production of these cytokines will greatly help us to understand the role of CD4+ CD25+ T cells as well as of CD4+ CD25 T cells in the induction of ACAID and may substantially contribute to the study on the strategies to prevent and treat autoimmune diseases.

Acknowledgements

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

This study was supported by Project of International Cooperation in Science and Technology, Guangdong Province (2006A50107001); the Key Project of National Natural Science Foundation (30630064); and the Project of Science and Technology of Guangdong Province (2005B60302009).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Wilbanks GA, Mammolenti M, Streilein JW. Studies on the induction of anterior chamber-associated immune deviation (ACAID). II. Eye-derived cells participate in generating blood-borne signals that induce ACAID. J Immunol 1991; 146:301824.
  • 2
    Wilbanks GA, Streilein JW. Studies on the induction of anterior chamber-associated immune deviation (ACAID). 1. Evidence that an antigen-specific, ACAID-inducing, cell-associated signal exists in the peripheral blood. J Immunol 1991; 146:26107.
  • 3
    Wilbanks GA, Mammolenti M, Streilein JW. Studies on the induction of anterior chamber-associated immune deviation (ACAID). Induction of ACAID depends upon intraocular transforming growth factor-β*. J Immunol 1992; 22:16573.
  • 4
    Wilbanks GA, Streilein JW. Macrophages capable of inducing anterior chamber associated immune deviation demonstrate spleen-seeking migratory properties. Reg Immunol 1992; 4:1307.
  • 5
    Hara Y, Caspi RR, Wiggert B, Dorf M, Streilein JW. Analysis of an in vitro-generated signal that induces systemic immune deviation similar to that elicited by antigen injected into the anterior chamber of the eye. J Immunol 1992; 149:15318. Erratum in: J Immunol 1992; 149:4116.
  • 6
    Wilbanks GA, Streilein JW. Fluids from immune privileged sites endow macrophages with the capacity to induce antigen-specific immune deviation via a mechanism involving transforming growth factor-β. Eur J Immunol 1992; 22:1031.
  • 7
    Hara Y, Okamoto S, Rouse B, Streilein JW. Evidence that peritoneal exudates cells cultured with eye-derived fluids are the proximate antigen-presenting cells in immune deviation of the ocular type. J Immunol 1993; 151:5162.
  • 8
    Takeuchi M, Kosiewicz MM, Alard P, Streilein JW. On the mechanisms by which transforming growth factor-β2 alters antigen-presenting abilities of macrophages on T cell activation. Eur J Immunol 1997; 27:164856.
  • 9
    Lan RY, Ansari AA, Lian ZX, Gershwin ME. Regulatory T cells: development, function and role in autoimmunity. Autoimmun Rev 2005; 4:35163.
  • 10
    Cobbold SP. T cell tolerance in transplantation: possibilities for therapeutic intervention. Expert Opin Ther Targets 2002; 6:58399. Review.
  • 11
    Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nat Immunol 2001; 2:81622. Review.
  • 12
    Read S, Powrie F. CD4 (+) regulatory T cells. Curr Opin Immunol 2001; 13:6449. Review.
  • 13
    Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, Bandeira A. On the ontogeny and physiology of regulatory T cells. Immunol Rev 2001; 182:517. Review.
  • 14
    Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol 2002; 2:389400.
  • 15
    Sakaguchi S, Sakaguchi N, Shimizu J et al. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001; 182:1832.
  • 16
    Kohm AP, Carpentier PA, Anger HA, Miller SD. Cutting edge: CD4+ CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol 2002; 169:47126.
  • 17
    Thorstenson KM, Khoruts A. Generation of anergic and potentially immunoregulatory CD25+ CD4+ T cells in vivo after induction of peripheral tolerance with intravenous or oral antigen. J Immunol 2001; 182:18895.
  • 18
    Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299:105761. Epub 2003 Jan 9.
  • 19
    Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+ CD25+ T regulatory cells. Nat Immunol 2003; 4:33742.
  • 20
    Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat Immunol 2003; 4:3306.
  • 21
    Hori S, Sakaguchi S. Foxp3: a critical regulator of the development and function of regulatory T cells. Microbes Infect 2004; 6:74551.
  • 22
    Sakaguchi S. Naturally arising Foxp3-expressing CD25+ CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005; 6:34552.
  • 23
    Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 2005; 22:32941.
  • 24
    Fantini MC, Becker C, Montelenone G, Pallone F, Galle PR, Neurath MF. Cutting edge: TGF-β induces a regulatory phenotype in CD4+ CD25 T cells through Foxp3 induction and down-regulation of smad7. J Immunol 2004; 172:514953.
  • 25
    Schramm C, Huber S, Protschka M et al. TGF-β regulates the CD4+ CD25+ T-cell pool and the expression of Foxp3 in vivo. Int Immunol 2004; 16:12419.
  • 26
    Park HB, Paik DJ, Jang E, Hong S, Youn J. Acquisition of anergic and suppressive activities in transforming growth factor-β-costimulated CD4+ CD25 T cells. Int Immunol 2004; 16:120313.
  • 27
    Chen W, Jin W, Harden N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+ CD25- Naïve T cells to CD4+ CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J Exp Med 2003, 198:187586.
  • 28
    Mahnke K, Schmitt E, Bonifaz L, Enk AH, Jonuleit H. Immature, but not inactive: the tolerogenic function of immature dendritic cells. Immunol Cell Biol 2002; 80:47783.
  • 29
    D’Orazio TJ, Niederkorn JY. A novel role for TGF-beta and IL-10 in the induction of immune privilege. J Immunol 1998; 160:208998.
  • 30
    Levings MK, Roncarolo MG. T-regulatory 1 cells: a novel subset of CD4 T cells with immunoregulatory properties. J Allergy Clin Immunol 2000; 106:s109.
  • 31
    Takahashi M, Ishimaru N, Yanagi K, Saegusa K, Haneji N, Shiota H, Hayashi Y. Requirement for splenic CD4+ T cells in the immune privilege of the anterior chamber of the eye. Clin Exp Immunol 1999; 116:2317.
  • 32
    Groux H. Type 1 T-regulatory cells: their role in the control of immune responses. Transplantation 2003; 75(Suppl. 9):8S.
  • 33
    Pyzik M, Piccirillo CA. TGF-beta1 modulates Foxp3 expression and regulatory activity in distinct CD4+ T cell subsets. J Leukoc Biol 2007; 82:33546. Epub 2007 May 2.
  • 34
    Classen S, Zander T, Eggle D et al. Human resting CD4+ T cells are constitutively inhibited by TGF beta under steady-state conditions. J Immunol 2007; 178:693140.
  • 35
    Seder RA, Marth T, Sieve MC, Strober W, Letterio JJ, Roberts AB, Kelsall B. Factors involved in the differentiation of TGF-beta-producing cells from naive CD4+ T cells: IL-4 and IFN-gamma have opposing effects, while TGF-beta positively regulates its own production. J Immunol 1998; 160:571928.
  • 36
    Marie JC, Letterio JJ, Gavin M, Rudensky AY. TGF-beta1 maintains suppressor function and Foxp3 expression in CD4+ CD25+ regulatory T cells. J Exp Med 2005; 201:10617.
  • 37
    Nakamura K, Kitani A, Fuss I, Pedersen A, Harada N, Nawata H, Strober W. TGF-beta 1 plays an important role in the mechanism of CD4+ CD25+ regulatory T cell activity in both humans and mice. J Immunol 2004; 172:83442.
  • 38
    Levings MK, Sangregorio R, Sartirana C, Moschin AL, Battaglia M, Orban PC, Roncarolo MG. Human CD25+ CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J Exp Med 2002; 196:133546.
  • 39
    Yamagiwa S, Gray JD, Hashimoto S, Horwitz DA. A role for TGF-β in the generation and expansion of CD4+ CD25+ regulatory T cells from human peripheral blood. J Immunol 2001; 166:7282.
  • 40
    Zheng SG, Gray JD, Ohtsuka K, Yamagiwa S, Horwitz DA. Generation ex vivo of TGF-β-producing regulatory T cells from CD4+ CD25 precursors. J Immunol 2002; 169:41839.
  • 41
    Zheng SG, Meng L, Wang JH, Watanabe M, Barr ML, Cramer DV, Gray JD, Horwitz DA. Transfer of regulatory T cells generated ex vivo modifies graft rejection through induction of tolerogenic CD4+ CD25+ cells in the recipient. Int Immunol 2006; 18:27989. Epub 2006 Jan 13.
  • 42
    Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+ CD25+ cells educate CD4+ CD25 cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10. J Immunol 2004; 172:521321.
  • 43
    Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, Enk AH. Infectious tolerance: human CD25+ regulatory T cells convey suppressor activity to conventional CD4+ T helper cells. J Exp Med 2002; 196:255.
  • 44
    Masli S, Turpie B, Hecker KH, Streilein JW. Expression of thrombospondin in TGF beta-treated APCs and its relevance to their immune deviation-promoting properties. J Immunol 2002; 168:226473.
  • 45
    Brunkow ME, Jeffery EW, Hjerrild KA et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 2001; 27:6873.
  • 46
    Jankovic D, Kullberg MC, Feng CG et al. Conventional T-bet (+) Foxp3 (−) Th1 cells are the major source of host-protective regulatory IL-10 during intracellular protozoan infection. J Exp Med 2007; 204:27383. Epub 2007 Feb 5.
  • 47
    Nicolson KS, O’Neill EJ, Sundstedt A, Streeter HB, Minaee S, Wraith DC. Antigen-induced IL-10+ regulatory T cells are independent of CD25+ regulatory cells for their growth, differentiation, and function. J Immunol 2006; 176:532937.
  • 48
    Baumgart M, Tompkins F, Leng J, Hesse M. Naturally occurring CD4+ Foxp3+ regulatory T cells are an essential, IL-10-independent part of the immunoregulatory network in Schistosoma mansoni egg-induced inflammation. J Immunol 2006; 176:537487.
  • 49
    Anderson CF, Oukka M, Kuchroo VJ, Sacks D. CD4 (+) CD25 (−) Foxp3 (−) Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis. J Exp Med 2007; 204:28597. Epub 2007 Feb 5.