January 2010
Volume 51, Issue 1
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Immunology and Microbiology  |   January 2010
Contribution of CD4+CD25+ T Cells to the Regression Phase of Experimental Autoimmune Uveoretinitis
Author Affiliations & Notes
  • Min Sun
    From the The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, People's Republic of China;
    Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People's Republic of China; and
  • Peizeng Yang
    From the The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, People's Republic of China;
  • Liping Du
    From the The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, People's Republic of China;
  • Hongyan Zhou
    Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People's Republic of China; and
  • Xiangrong Ren
    Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People's Republic of China; and
  • Aize Kijlstra
    Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital Maastricht, Maastricht, The Netherlands.
  • Corresponding author: Peizeng Yang, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing 400016, P. R. China; peizengycmu@126.com
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 383-389. doi:10.1167/iovs.09-3514
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      Min Sun, Peizeng Yang, Liping Du, Hongyan Zhou, Xiangrong Ren, Aize Kijlstra; Contribution of CD4+CD25+ T Cells to the Regression Phase of Experimental Autoimmune Uveoretinitis. Invest. Ophthalmol. Vis. Sci. 2010;51(1):383-389. doi: 10.1167/iovs.09-3514.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate the role of CD4+CD25+ Treg cells in the development of experimental autoimmune uveoretinitis (EAU).

Methods.: EAU was induced in B10RIII mice by immunization with IRBP161–180 in complete Freund's adjuvant and evaluated clinically and pathologically on days 0, 7, 14, 21, and 28. Lymphocytes from draining lymph nodes (LNs) were subjected to flow cytometry to analyze the frequency of CD4+CD25+ Treg cells. CD4+CD25+ Treg cells and CD4+CD25 T cells were separated by means of magnetic-assisted cell sorting and cocultured or crossover cultured for 3 days. Proliferation of CD4+CD25 T cells was measured using a modified MTT assay. The levels of IFN-γ and IL-17 in the supernatants were determined by enzyme-linked immunosorbent assay.

Results.: Clinical and histopathologic results showed a severe intraocular inflammation in the immunized mice. The frequency of CD4+Foxp3+ T cells and CD4+CD25+Foxp3+ T cells in the draining LN lymphocytes was increased on day 7, reached its peak on day 14, and maintained a high level up to day 42. CD4+CD25+ Treg cells obtained from mice on days 14 and 28 after immunization showed a stronger inhibitory effect on the proliferation of CD4+CD25 T cells and the production of IFN-γ by CD4+CD25 T cells compared with those obtained from control mice. CD4+CD25+ Treg cells did not affect IL-17 production. Transfer of CD4+CD25+ Treg cells obtained from EAU mice was able to suppress EAU induction by IRBP161–180 that was not observed after transfer of cells from mice that had received CFA alone, suggesting antigen specificity of the Treg response.

Conclusions.: A significantly increased frequency and immunoregulatory action of CD4+CD25+ Treg cells is associated with the development and regression of EAU, suggesting that CD4+CD25+ Treg cells are induced during EAU and may be involved in its regression.

CD4+CD25+ regulatory T cells (Treg cells), a functionally distinct subpopulation of T cells differentiated in the normal thymus, play a critical role in the maintenance of peripheral self-tolerance throughout a person's lifespan. 1,2 Functional abrogation or deficiency of CD4+CD25+ Treg cells has been shown to be responsible for the development of various autoimmune diseases in rodents and humans. 3 CD4+CD25+ Treg cells specifically express Foxp3, a transcription factor that is generally considered a specific molecular marker for CD4+CD25+ Treg cells in rodents and humans. 4 In the absence of Foxp3-expressing CD4+CD25+ Treg cells, autoreactive T-cell clones may become activated and induce severe and widespread autoimmune disease. 5,6  
Experimental autoimmune uveoretinitis (EAU), an organ-specific autoimmune disease model, has been widely used as a model for human intraocular inflammation. 7 This model can be induced in mice by immunization with retinal antigens such as interphotoreceptor retinoid-binding protein (IRBP), the soluble retinal antigen (S-antigen), 8 or their polypeptides. Histopathologically, EAU is similar to human uveitis and is characterized by posterior retinal and choroidal lesions, granuloma formation, vasculitis, photoreceptor damage, vitritis, and varying degrees of inflammatory infiltration in the anterior segment of the eye. 9 A number of groups have recently shown that both Th1- and Th17- effector T cells (Teff cells) play an important role in the development and maintenance of EAU. 10,11 EAU differs from human uveitis in that the model has a striking monophasic nature, whereas human disease often presents as a chronic relapsing condition. Treg cells may evolve during EAU and offer an explanation for its monophasic nature. 12  
The role of CD4+CD25+ Treg cells in autoimmune disease has been reported in animal models of experimental autoimmune encephalomyelitis (EAE) 13,14 and arthritis. 15 Others have demonstrated that hydrodynamic vaccination with DNA encoding IRBP could protect from autoimmunity through the induction of Treg cells. 16 Natural Treg cells and Treg cells induced by LPS-activated bone marrow dendritic cells could suppress the development of EAU. 17,18 However, little is known about the role of CD4+CD25+ Treg cells and the relationship between CD4+CD25+ Treg cells and Teff cells in EAU. In the present study, therefore, we investigated the frequency and function of CD4+CD25+ Treg cells in EAU induced by IRBP161–180 in B10RIII mice. Our results showed an increased frequency of CD4+Foxp3+ T cells and CD4+CD25+Foxp3+ T cells (CD4+CD25+ Treg cells) during EAU. Furthermore, we found that these Treg cells showed a strong inhibitory effect on the proliferation of CD4+CD25 T cells and on the production of IFN-γ by CD4+CD25 T cells. These results suggest that CD4+CD25+ Treg cells may play an important role in the recovery phase of EAU. 
Materials and Methods
Mice and Reagents
B10RIII mice (6–8 weeks of age) were purchased from Jackson Laboratory (Bar Harbor, ME) and were housed under standard (specific pathogen-free) conditions. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. IRBP161–180 (SGIPYIISYLHPGNTILHVD) was synthesized by Shanghai Sangon Biological Engineering Technology & Services Ltd. Co. Complete Freund's adjuvant (CFA) containing 1.0 mg/mL Mycobacterium tuberculosis and pertussis toxin (PTX) were obtained from Sigma-Aldrich (St. Louis, MO). 
Immunization
For induction of EAU, mice (8–12 weeks of age) were immunized subcutaneously with a 200 μL emulsion containing 50 μg IRBP161–180 in CFA. PTX (1.0 μg) was concurrently injected intraperitoneally as an additional adjuvant. Control groups of mice received an emulsion of 50 μL PBS and 150 μL CFA that was injected subcutaneously. 
Clinical Examination and Histopathologic Evaluation
After immunization, the animals were observed daily by slit lamp microscopy and ophthalmoscopy starting on day 7 until day 28. Eyes were enucleated from control and immunized mice on days 7, 14, 21, and 28 after IRBP immunization and were fixed for 1 hour in 4% buffered glutaraldehyde and then transferred to 10% buffered formaldehyde until processing. Fixed and dehydrated tissue was embedded in paraffin, and 4- to 6-μm sections were stained by standard hematoxylin and eosin (H&E). The intensity of EAU was scored in a masked fashion from 0 to 4 according to the histopathologic grading described previously for murine EAU. 19  
Flow Cytometry
Draining lymph nodes (LNs; inguinal and iliac) were dissected and ground from control mice and those after IRBP immunization on days 7, 14, 21, 28, and 42. The obtained lymphocytes were passed through a sterile wire mesh and stained by direct immunofluorescence using four-color flow cytometry (FCM; FACSAire; Becton Dickinson, Franklin Lakes, NJ). To analyze cell surface molecule expression, aliquots of 1 × 106 cells were stained with combinations of FITC-, PE-cy7, or PE-conjugated mAbs against mouse CD3, CD4, or CD25 (eBioscience; San Diego, CA) for 30 minutes, followed by fixation in 4% paraformaldehyde. Alternatively, cells were permeabilized and fixed using fixation/permeabilization solution (eBioscience) and were stained with the APC-conjugated Foxp3 mAbs (eBioscience) for 1 hour and were finally subjected to FCM analysis. Six mice were used in each experimental group. These experiments were repeated three times. 
Preparation of Peritoneal Exudate Cells
Intraperitoneal injection of thioglycolate (Sigma-Aldrich) was performed, and peritoneal exudate cells were harvested 3 days later. As described previously, 20 the recovered cells were washed and resuspended, placed in a 24-well culture plate (1 × 106/well), and incubated in complete RPMI 1640 medium at 37°C, 5% CO2. After overnight culture, plates were washed three times with culture medium to remove nonadherent cells. Adherent cells were retained in the wells and used as antigen-presenting cells (APCs). More than 90% of these adherent cells were F4/80+, as identified by subsequent FCM analysis. 
Cell Purification of CD4+CD25+ T Cells
At day 14 or 28 after IRBP immunization, CD4+CD25+ T cells were isolated from mouse lymphocytes obtained from draining LNs with a CD4+CD25+ regulatory T cells isolation kit (Miltenyi Biotec, Palo Alto, CA). Briefly, lymphocyte suspensions were incubated with a cocktail of biotin-conjugated antibodies and anti-biotin microbeads for negative sorting of CD4+ T cells. CD4+ T cells were then separated into two aliquots with adequate cell numbers according to the requirements of the experiment. One aliquot was used to separate CD4+CD25 T cells by the addition of a recommended volume of anti-CD25 beads according to the manufacturer's instructions. The purity of isolated CD4+CD25 T cells, as identified by FCM analysis, was shown to be higher than 98%. The other aliquot was used to separate CD4+CD25+ Treg cells using a reduced volume of anti-CD25 beads. To obtain a highly purified CD4+CD25+ cell population, we reduced the recommended volume of anti-CD25 beads to a concentration of 1 μL per 1 × 107 lymphocytes. The purity of isolated CD4+CD25+ T cells identified by FCM analysis was higher than 96%. Therefore, this concentration of anti-CD25 beads was used for the isolation of CD4+CD25+ Treg cells from both EAU and control groups of mice and were used in the experiments as described. 
In Vitro Cell Stimulation and Suppressor Assay
To test the inhibition of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells, a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a cell counting kit (Cell Counting Kit-8; Sigma-Aldrich, St. Louis, MO) was performed to detect cell proliferation, as previously described. 21 In the presence of peritoneal exudate cells (1 × 105) as APCs, 2.0 × 104 CD4+CD25 T cells were cultured with or without various numbers of CD4+CD25+ Treg cells at 37°C and 5% CO2 for 3 days in 96-well round-bottomed plates in 200 μL of 1640 RPMI medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. For crossover experiments, 2 × 104 CD4+CD25 T cells from control groups in the presence of 1 × 105 APCs were mixed at a ratio of 1:1 with CD4+CD25+ Treg cells from EAU mice, and vice versa. Anti-CD3 mAb (BD PharMingen, Franklin Lakes, NJ) at a final concentration of 1 μg/mL was added to the culture for stimulation. Eventually, 10 μL WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) was added and incubated for 4 hours. Absorbance was determined at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek Instruments, Winooski, VT). Two mice were used for each group in one experiment. The experiment was repeated at least three times. 
ELISA
To analyze the secretion of cytokines, 200 μL supernatant was removed from the cultures of the proliferation and crossover experiments. Levels of IFN-γ and IL-17 were measured using commercially available ELISA kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN) with detection limits of 30 pg/mL and 15 pg/mL, respectively. 
Adoptive Transfer
To further identify the inhibitory function of Treg cells, adoptive transfer experiments were performed. CD4+CD25+ Treg cells were sorted and resuspended in physiological phosphate-buffered saline (PBS) from draining LNs of control mice and immunized groups on day 14 or 28. Cells (5.0 × 104) were injected intravenously into naive syngeneic mice on the day of immunization with IRBP161–180 to induce EAU. On day 14, eyes were enucleated from each group and stained by standard H&E, as described. The intensity of EAU was scored in a masked fashion from 0 to 4 according to the histopathologic grading. 
Statistical Analysis
Data were expressed as the mean ± SD. Groups were compared with one-way analysis of variance, and P < 0.05 was taken as a statistically significant difference. Analysis was performed using software (SPSS version 13.0; SPSS, Chicago, IL). 
Results
Induction of EAU
EAU was successfully induced in B10RIII mice after immunization with 50 μg IRBP161–180 in CFA and was characterized clinically by aqueous flare, cells, exudates, and severe changes in the retina and uvea (data not shown). Histologic examination showed a severe inflammation in the anterior segment and a massive influx of mononuclear and polymorphonuclear cells into the retina and choroid, granuloma formation, vasculitis, photoreceptor loss, and vitritis (Fig. 1). This inflammation was already evident on day 8 to 9 and reached its peak by day 14, followed by a rapid regression. In line with published reports, there was no inflammation in normal mice or control mice immunized with CFA only 19 (Fig. 1). 
Figure 1.
 
EAU induced in B10RIII mice by immunization with 50 μg IRBP161–180 in CFA. (A) Normal retinal structure in B10RIII mouse receiving CFA alone. Hematoxylin-eosin, magnification ×200. (B) Image from day 14 after immunization (at the peak of inflammation) shows inflammatory infiltrating cells (filled arrow) in the vitreous, the retina, and the choroid, vasculitis (open arrow), and damage to the retinal photoreceptor cell layer (asterisk). Hematoxylin-eosin, magnification ×200. (C) Image from day 21 after immunization shows no obvious inflammation in the retina except some minor scarring caused by the previous damage. Hematoxylin-eosin, magnification ×200. (D) Mean histopathologic score during the development of EAU. Six mice were used in each group.
Figure 1.
 
EAU induced in B10RIII mice by immunization with 50 μg IRBP161–180 in CFA. (A) Normal retinal structure in B10RIII mouse receiving CFA alone. Hematoxylin-eosin, magnification ×200. (B) Image from day 14 after immunization (at the peak of inflammation) shows inflammatory infiltrating cells (filled arrow) in the vitreous, the retina, and the choroid, vasculitis (open arrow), and damage to the retinal photoreceptor cell layer (asterisk). Hematoxylin-eosin, magnification ×200. (C) Image from day 21 after immunization shows no obvious inflammation in the retina except some minor scarring caused by the previous damage. Hematoxylin-eosin, magnification ×200. (D) Mean histopathologic score during the development of EAU. Six mice were used in each group.
Kinetics of CD4+Foxp3+ T Cells in EAU
The frequency of CD4+Foxp3+ T cells in the lymphocytes from LNs of normal mice and those obtained at different time points after immunization with IRBP was assessed using flow cytometry. As shown in Figure 2A, an increased frequency of CD4+Foxp3+ T cells was noted on day 7 (12.7%). The highest frequency of these cells was observed on day 14 (20.7%) and was followed by a slight decrease on days 21 (18.3%), 28 (19.6%), and 42 (18.9%), though the decrease failed to show a statistically significant difference. The frequency of CD4+Foxp3+ T cells in the control mice that had received CFA alone remained unchanged during the experiment. 
Figure 2.
 
Flow cytometric assay of Foxp3 expression on draining LN CD4+ T cells (A) or CD4+CD25+ T cells (B) from mice after immunization with IRBP peptide on days 0, 7, 14, 21, 28, and 42 or in control groups receiving CFA alone. Lymphocytes were harvested, stained with anti-CD3, anti-CD4, anti-CD25, anti-Foxp3, or isotype-matched antibodies, and assessed for Foxp3 on CD4+ T cells or CD4+CD25+ T cells. Bars show the mean ± SD frequency of each group. Six animals were used in each experimental group.
Figure 2.
 
Flow cytometric assay of Foxp3 expression on draining LN CD4+ T cells (A) or CD4+CD25+ T cells (B) from mice after immunization with IRBP peptide on days 0, 7, 14, 21, 28, and 42 or in control groups receiving CFA alone. Lymphocytes were harvested, stained with anti-CD3, anti-CD4, anti-CD25, anti-Foxp3, or isotype-matched antibodies, and assessed for Foxp3 on CD4+ T cells or CD4+CD25+ T cells. Bars show the mean ± SD frequency of each group. Six animals were used in each experimental group.
Kinetics of CD4+CD25+ Foxp3+ T Cells in EAU
We further analyzed the frequency of CD4+CD25+Foxp3+ T cells in LN lymphocytes. A dynamic change in the frequency of LN CD4+CD25+Foxp3+ T cells was observed after immunization at different time points. The frequency of CD4+CD25+Foxp3+ T cells was increased on day 7 (6.47%) and reached its peak on day 14 (13.25%). A high frequency of CD4+CD25+Foxp3+ T cell was still observed in the LNs on days 21 (9.25%), 28 (10.94%), and 42 (9.90%). No change in the frequency of CD4+CD25+Foxp3+ T cells from day 0 to day 42 was noted in the CFA control mice (Fig. 2B). 
Inhibition of CD4+CD25+ Treg Cells on the Proliferation of CD4+CD25 T Cells in EAU
As shown in Figures 3A and 3B, the purities of CD4+CD25+ T cells or CD4+CD25 Teff cells sorted by magnetic beads were greater than 96% or 98%, respectively, and most of the CD4+CD25+ T cells (>96%) expressed Foxp3 in all groups analyzed. These results confirmed that almost all the CD4+CD25+ T cells were Treg cells. A proliferation assay using anti-CD3 antibodies as a stimulus was performed to evaluate the suppressive potential of these Treg cells. The results revealed that, at a ratio of 1:1, CD4+CD25+ Treg cells isolated from EAU mice on day 14 or 28 after immunization suppressed CD4+CD25 T-cell proliferation by an average of 89.3% or 64.0%, respectively, which was significantly higher than that observed when using cells obtained from control mice (44.2%) (day 14, P < 0.001; day 28, P = 0.027) (Fig. 3C). 
Figure 3.
 
Inhibitory effect of CD4+CD25+ T cells on the proliferation of CD4+CD25 T cells on anti-CD3 mAb stimulation. Proliferation of cultured cells was tested by a modified MTT assay. Results are expressed as mean ± SD. (A, B) Representative flow cytometry diagrams illustrating the frequency of CD4+CD25± T cells and the percentage of Foxp3 on CD4+CD25± (at the peak of inflammation) isolated by MACS. (C) The suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 on anti-CD3 stimulation. (D, E) Crossover experiments to further identify the suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 at the inflammatory or recovery phase of EAU. (F) Comparison of the relative inhibitory activity of CD4+CD25+ Treg cells from immunized mice and control groups on the proliferation of CD4+CD25 T cells at various ratios of these two cell populations.
Figure 3.
 
Inhibitory effect of CD4+CD25+ T cells on the proliferation of CD4+CD25 T cells on anti-CD3 mAb stimulation. Proliferation of cultured cells was tested by a modified MTT assay. Results are expressed as mean ± SD. (A, B) Representative flow cytometry diagrams illustrating the frequency of CD4+CD25± T cells and the percentage of Foxp3 on CD4+CD25± (at the peak of inflammation) isolated by MACS. (C) The suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 on anti-CD3 stimulation. (D, E) Crossover experiments to further identify the suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 at the inflammatory or recovery phase of EAU. (F) Comparison of the relative inhibitory activity of CD4+CD25+ Treg cells from immunized mice and control groups on the proliferation of CD4+CD25 T cells at various ratios of these two cell populations.
A crossover experiment was performed to further identify the increased regulatory activity of the CD4+CD25+ Treg cells in EAU mice. The inhibitory effect of CD4+CD25+ Treg cells from the EAU mice on day 14 or 28 showed enhanced inhibition on the proliferation of CD4+CD25 T cells of control groups compared with Treg cells obtained from control mice (P < 0.001, P = 0.023, respectively) (Figs. 3D, 3E). 
In addition, we quantified the inhibitory function of CD4+CD25+ Treg cells using various cell-to-cell ratios. At the same ratio of Treg cells to Teff cells, such as 1:1 or 1:2, the suppressive capacity of Treg cells from immunized mice was higher than that from control groups. Moreover, the Treg cells from the immunized mice on day 14 were stronger in suppressing Teff cells than those from immunized mice on day 28. In all these three groups, by decreasing the ratio of CD4+CD25+ Treg cells to CD4+CD25 Teff cells, the inhibitory ability of CD4+CD25+ Treg cells was gradually diminished. At a ratio of 1:8 to 1:16, CD4+CD25+ Treg cells were unable to inhibit the proliferation of CD4+CD25 T cells (Fig. 3F). 
Influence of CD4+CD25+ T Cells on the Production of IFN-γ and IL-17
Given that both Th1 cells and Th17 cells play an important role in the development of EAU, we investigated whether the CD4+CD25+ Treg cells exerted their regulatory function on CD4+CD25 T cells through the modulation of IFN-γ and IL-17. These cytokines are considered to be representative for Th1 and Th17 cells, respectively. 
Results of the ELISA on supernatants obtained from the proliferation experiments described showed a significantly increased IFN-γ level on day 14 (day 14 vs control, P < 0.001) but a lower level of this cytokine on day 28 (day 28 vs control, P = 0.032). When cultured with CD4+CD25+ Treg cells, IFN-γ production by CD4+CD25 T cells was significantly decreased both in control groups and in the EAU mice on days 14 and 28 after immunization. Stronger inhibition was noted in the immunized mice than in control groups (day 14, P < 0.001; day 28, P = 0.001; Fig. 4A). Moreover, the crossover experiments showed that inhibition of IFN-γ production by CD4+CD25+ Treg cells from immunized mice was stronger than that seen when using Treg cells from control mice (Figs. 4B, 4C). 
Figure 4.
 
Inhibitory effect of CD4+CD25+ Treg cells on the production of IFN-γ and IL-17 by cocultured CD4+CD25 T cells on stimulation with anti-CD3 mAb. Levels of IFN-γ from cultured supernatants were determined by ELISA. All data were expressed as mean ± SD. (A) CD4+CD25+ Treg cells from immunized mice and control groups were able to inhibit the production of IFN-γ secreted by the cocultured CD4+CD25 T cells. CD4+CD25+ Treg cells from immunized mice had enhanced inhibitory capacity compared with these cells from control groups. (B, C) CD4+CD25 T cells from immunized mice in the presence of APCs were cocultured with CD4+CD25+ Treg cells from control groups, and vice versa. CD4+CD25+ Treg cells from immunized mice were more powerful in inhibiting the production of IFN-γ. (DF) CD4+CD25+ Treg cells from immunized mice or control groups did not affect the secretion of IL-17 by CD4+CD25 T cells. ND, not detected.
Figure 4.
 
Inhibitory effect of CD4+CD25+ Treg cells on the production of IFN-γ and IL-17 by cocultured CD4+CD25 T cells on stimulation with anti-CD3 mAb. Levels of IFN-γ from cultured supernatants were determined by ELISA. All data were expressed as mean ± SD. (A) CD4+CD25+ Treg cells from immunized mice and control groups were able to inhibit the production of IFN-γ secreted by the cocultured CD4+CD25 T cells. CD4+CD25+ Treg cells from immunized mice had enhanced inhibitory capacity compared with these cells from control groups. (B, C) CD4+CD25 T cells from immunized mice in the presence of APCs were cocultured with CD4+CD25+ Treg cells from control groups, and vice versa. CD4+CD25+ Treg cells from immunized mice were more powerful in inhibiting the production of IFN-γ. (DF) CD4+CD25+ Treg cells from immunized mice or control groups did not affect the secretion of IL-17 by CD4+CD25 T cells. ND, not detected.
Significantly higher production of IL-17 by CD4+CD25 T cells was observed in the mice after immunization on day 14, compared with control groups (P < 0.001). Increased production of this cytokine, though lower than that observed in the mice on day 14, was also noted in the mice on day 28 (P < 0.001). When these cells were cocultured with CD4+CD25+ Treg cells either from immunized mice or from control groups, we did not observe a significant change in the production of IL-17 by CD4+CD25 T cells (Figs. 4D–4F). 
Inhibitory Function of Treg Cells In Vivo
As shown in Figure 5, transfer of CD4+CD25+ Treg cells obtained from immunized mice on day 14 or 28 could suppress the activity of EAU induced by IRBP161–180 (day 14, P < 0.001; day 28, P = 0.004). Transfer of Treg cells obtained from control groups showed a slight decrease in the intensity of EAU that did not reach statistical difference when compared with the immunized mice that had not received cells (P = 0.235). 
Figure 5.
 
Adoptive transfer experiment to demonstrate the function of Treg cells in vivo. CD4+CD25+ Treg cells from control mice and immunized mice on days 14 and 28 were isolated and injected intravenously into naive syngeneic mice on the day of immunization with IRBP161–180 to induce EAU. The intensity of EAU on day 14 was scored in a masked fashion from 0 to 4 according to histopathologic grading. Transfer of CD4+CD25+ Treg cells from immunized mice obtained on day 14 or 28 could suppress EAU. Four mice were used in each group.
Figure 5.
 
Adoptive transfer experiment to demonstrate the function of Treg cells in vivo. CD4+CD25+ Treg cells from control mice and immunized mice on days 14 and 28 were isolated and injected intravenously into naive syngeneic mice on the day of immunization with IRBP161–180 to induce EAU. The intensity of EAU on day 14 was scored in a masked fashion from 0 to 4 according to histopathologic grading. Transfer of CD4+CD25+ Treg cells from immunized mice obtained on day 14 or 28 could suppress EAU. Four mice were used in each group.
Discussion
In this study we investigated the frequency and function of CD4+CD25+ Treg cells in EAU induced by IRBP161–180 in B10RIII mice. The results showed an increased frequency of CD4+CD25+ Treg cells in association with the EAU activity. These Treg cells were able to inhibit proliferation and IFN-γ production by CD4+CD25 target cells, an activity that increased during the development of the EAU. This association of increased frequency and strengthened function of CD4+CD25+ Treg cells with the regression of EAU activity suggests that these cells may contribute to the short-lived intraocular autoimmune inflammation induced by IRBP-peptide in mice. Adoptive transfer experiments we performed whereby CD4+CD25+ Treg cells obtained from EAU mice were injected into naive animals that were subsequently challenged with IRBP161–180 in the presence of CFA showed that EAU induction could be suppressed. This was not observed after transfer of Treg cells from mice that had received CFA alone, therefore suggesting antigen specificity of the Treg response. We did not inject additional pertussis in these control groups; further experiments are necessary to evaluate a possible role of pertussis in the induction of Tregs. Additional experiments are needed using immunizations with other autoantigens (e.g., myelin oligodendrocyte glycoprotein peptide) to prove the ocular autoantigen specificity of the induced Treg response in our model. Our data are consistent with earlier results showing that adoptively transferred Treg cells specific for islet antigens were more potent at blocking type 1 diabetes than polyclonal Treg cells. 22  
A recent study in our laboratory showed decreased frequency and impaired function of CD4+CD25+ Treg cells in VogtKoyanagi-Harada (VKH) syndrome. 23 In the study presented here, we investigated whether this could also be shown in an experimental uveitis model whereby EAU was induced in B10RIII mice by immunization with IRBP161–180. Clinical examination and histopathologic evaluation showed an intraocular inflammation similar to human uveitis, thereby providing a good model for investigating the role of Treg cells in uveitis. We focused on CD4+ Treg cells, which have been shown to be critical for the development of autoimmune disease. 24 Because Foxp3 and CD25 are important makers for CD4+ Treg cells, we investigated the frequency of Foxp3-expressing CD4+ T cells and of Foxp3-expressing CD4+CD25+ T cells. Results showed a significantly increased frequency of these two populations, as was also demonstrated earlier in a murine model of systemic lupus erythematosus (SLE). 25 Our results confirmed that the CD4+CD25+ Treg cell subpopulation was expanded in mice undergoing EAU induced by IRBP161–180. It is interesting to analyze the relationship between EAU ontogeny and the CD4+CD25+ Treg cells. Increased frequency of these Treg cells occurred on day 7 after immunization with IRBP161–180, a phase of EAU induction, suggesting that the induction of these Treg cells is closely associated with the development of EAU. The highest frequency of these Treg cells was observed on day 14 after immunization, a peak stage of intraocular inflammation in this model. Rapid resolution of the intraocular inflammation was noted thereafter in association with prolonged maintenance of a higher frequency of CD4+CD25+ Treg cells. These concurring epiphenomena of Treg cells and the regression of EAU suggest a possible causative relationship between these two events. A functional study was performed to test this presumption. Coculture experiments showed that CD4+CD25+ T cells could significantly inhibit the proliferation of CD4+CD25 T cells either in immunized mice or in control groups, confirming the Treg property of these cells. A crossover experiment was performed to compare the inhibitory effect of CD4+CD25+ T cells from the EAU mice with Treg cells from control mice on the proliferation of CD4+CD25 T cells. Interestingly, CD4+CD25+ T cells from immunized mice showed a stronger inhibitory effect than those obtained from control groups. A similar effect was shown concerning the capacity of Treg cells from EAU mice on CD4+CD25 T-cell production of IFN-γ. The inhibitory effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 Teff cells or the production of IFN-γ was also demonstrated in animal models of SLE or EAE, respectively. 25,26 Taken together, it is likely that CD4+CD25+ Treg cells with potent inhibitory properties are induced after immunization and that these Treg cells may be responsible for the rapid regression observed in this EAU model. 
It has been reported that both IFN-γ and IL-17 are involved in the development of certain autoimmune diseases, including uveitis. 27,28 Significantly increased production of these two cytokines was observed at the peak stage of EAU in our study. This result is consistent with data obtained in our laboratory in patients with VKH syndrome. 29,30 Interestingly, CD4+CD25+ Treg cells either from immunized mice or from control groups did not have a detectable effect on IL-17 production by CD4+CD25 T cells, confirming other results that Th17 cells, unlike Th1 cells, may not be suppressed by the population of Treg cells. 3134 The exact mechanisms underlying the resistance of Th17 cells to regulation by Treg cells has not yet been clarified and deserves further study. 
When comparing the CD4+ Treg cells in human VKH syndrome with those in EAU, an interesting difference can be noted. The frequency and function of Treg cells was decreased in VKH syndrome, contrary to the increased frequency and enhanced function of these Treg cells in EAU. These conflicting results between VKH syndrome and the EAU model may imply different mechanisms underlying human uveitis and its animal models. VKH syndrome, typically showing a chronic intraocular inflammation with recurrent episodes, is presumed to be caused by an immune disturbance possibly through dysfunctional Treg cells and may explain why the intraocular inflammation in these patients is not able to resolve with time. Unlike VKH syndrome, EAU is induced in mice with a normal immune regulatory capacity. It may, therefore, not be surprising that the intraocular inflammation induced by a break in the tolerance against certain retinal peptides, as seen in EAU, disappears rapidly after an upregulated immunoregulatory control mechanism elaborated by an increased frequency and enhanced function of Treg cells. Our findings confirm a recent study whereby the dynamics of Treg cells were studied in the retinas of mice undergoing IRBP-induced EAU. 12  
In conclusion, CD4+ Treg cells with strong immunoregulatory properties were induced in mice immunized with IRBP161–180; the emergence of this population was associated with a rapid resolution of EAU. These induced Treg cells may be, at least partially, responsible for the monophasic nature and rapid regression of EAU. 
Footnotes
 Supported by the Key Project of Natural Science Foundation (30630064), National Supporting Project of the People's Republic of China (2007BAI18B10), Key Project of Chongqing Health Bureau (2008–01-15), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), Key Project of Chongqing National Science Foundation (CSTC, 2009BA5037), and the Clinical Key Project of Ministry of Health.
Footnotes
 Disclosure: M. Sun, None; P. Yang, None; L. Du, None; H. Zhou, None; X. Ren, None; A. Kijlstra, None
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Figure 1.
 
EAU induced in B10RIII mice by immunization with 50 μg IRBP161–180 in CFA. (A) Normal retinal structure in B10RIII mouse receiving CFA alone. Hematoxylin-eosin, magnification ×200. (B) Image from day 14 after immunization (at the peak of inflammation) shows inflammatory infiltrating cells (filled arrow) in the vitreous, the retina, and the choroid, vasculitis (open arrow), and damage to the retinal photoreceptor cell layer (asterisk). Hematoxylin-eosin, magnification ×200. (C) Image from day 21 after immunization shows no obvious inflammation in the retina except some minor scarring caused by the previous damage. Hematoxylin-eosin, magnification ×200. (D) Mean histopathologic score during the development of EAU. Six mice were used in each group.
Figure 1.
 
EAU induced in B10RIII mice by immunization with 50 μg IRBP161–180 in CFA. (A) Normal retinal structure in B10RIII mouse receiving CFA alone. Hematoxylin-eosin, magnification ×200. (B) Image from day 14 after immunization (at the peak of inflammation) shows inflammatory infiltrating cells (filled arrow) in the vitreous, the retina, and the choroid, vasculitis (open arrow), and damage to the retinal photoreceptor cell layer (asterisk). Hematoxylin-eosin, magnification ×200. (C) Image from day 21 after immunization shows no obvious inflammation in the retina except some minor scarring caused by the previous damage. Hematoxylin-eosin, magnification ×200. (D) Mean histopathologic score during the development of EAU. Six mice were used in each group.
Figure 2.
 
Flow cytometric assay of Foxp3 expression on draining LN CD4+ T cells (A) or CD4+CD25+ T cells (B) from mice after immunization with IRBP peptide on days 0, 7, 14, 21, 28, and 42 or in control groups receiving CFA alone. Lymphocytes were harvested, stained with anti-CD3, anti-CD4, anti-CD25, anti-Foxp3, or isotype-matched antibodies, and assessed for Foxp3 on CD4+ T cells or CD4+CD25+ T cells. Bars show the mean ± SD frequency of each group. Six animals were used in each experimental group.
Figure 2.
 
Flow cytometric assay of Foxp3 expression on draining LN CD4+ T cells (A) or CD4+CD25+ T cells (B) from mice after immunization with IRBP peptide on days 0, 7, 14, 21, 28, and 42 or in control groups receiving CFA alone. Lymphocytes were harvested, stained with anti-CD3, anti-CD4, anti-CD25, anti-Foxp3, or isotype-matched antibodies, and assessed for Foxp3 on CD4+ T cells or CD4+CD25+ T cells. Bars show the mean ± SD frequency of each group. Six animals were used in each experimental group.
Figure 3.
 
Inhibitory effect of CD4+CD25+ T cells on the proliferation of CD4+CD25 T cells on anti-CD3 mAb stimulation. Proliferation of cultured cells was tested by a modified MTT assay. Results are expressed as mean ± SD. (A, B) Representative flow cytometry diagrams illustrating the frequency of CD4+CD25± T cells and the percentage of Foxp3 on CD4+CD25± (at the peak of inflammation) isolated by MACS. (C) The suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 on anti-CD3 stimulation. (D, E) Crossover experiments to further identify the suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 at the inflammatory or recovery phase of EAU. (F) Comparison of the relative inhibitory activity of CD4+CD25+ Treg cells from immunized mice and control groups on the proliferation of CD4+CD25 T cells at various ratios of these two cell populations.
Figure 3.
 
Inhibitory effect of CD4+CD25+ T cells on the proliferation of CD4+CD25 T cells on anti-CD3 mAb stimulation. Proliferation of cultured cells was tested by a modified MTT assay. Results are expressed as mean ± SD. (A, B) Representative flow cytometry diagrams illustrating the frequency of CD4+CD25± T cells and the percentage of Foxp3 on CD4+CD25± (at the peak of inflammation) isolated by MACS. (C) The suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 on anti-CD3 stimulation. (D, E) Crossover experiments to further identify the suppressive effect of CD4+CD25+ Treg cells on the proliferation of CD4+CD25 T cells at a ratio of 1:1 at the inflammatory or recovery phase of EAU. (F) Comparison of the relative inhibitory activity of CD4+CD25+ Treg cells from immunized mice and control groups on the proliferation of CD4+CD25 T cells at various ratios of these two cell populations.
Figure 4.
 
Inhibitory effect of CD4+CD25+ Treg cells on the production of IFN-γ and IL-17 by cocultured CD4+CD25 T cells on stimulation with anti-CD3 mAb. Levels of IFN-γ from cultured supernatants were determined by ELISA. All data were expressed as mean ± SD. (A) CD4+CD25+ Treg cells from immunized mice and control groups were able to inhibit the production of IFN-γ secreted by the cocultured CD4+CD25 T cells. CD4+CD25+ Treg cells from immunized mice had enhanced inhibitory capacity compared with these cells from control groups. (B, C) CD4+CD25 T cells from immunized mice in the presence of APCs were cocultured with CD4+CD25+ Treg cells from control groups, and vice versa. CD4+CD25+ Treg cells from immunized mice were more powerful in inhibiting the production of IFN-γ. (DF) CD4+CD25+ Treg cells from immunized mice or control groups did not affect the secretion of IL-17 by CD4+CD25 T cells. ND, not detected.
Figure 4.
 
Inhibitory effect of CD4+CD25+ Treg cells on the production of IFN-γ and IL-17 by cocultured CD4+CD25 T cells on stimulation with anti-CD3 mAb. Levels of IFN-γ from cultured supernatants were determined by ELISA. All data were expressed as mean ± SD. (A) CD4+CD25+ Treg cells from immunized mice and control groups were able to inhibit the production of IFN-γ secreted by the cocultured CD4+CD25 T cells. CD4+CD25+ Treg cells from immunized mice had enhanced inhibitory capacity compared with these cells from control groups. (B, C) CD4+CD25 T cells from immunized mice in the presence of APCs were cocultured with CD4+CD25+ Treg cells from control groups, and vice versa. CD4+CD25+ Treg cells from immunized mice were more powerful in inhibiting the production of IFN-γ. (DF) CD4+CD25+ Treg cells from immunized mice or control groups did not affect the secretion of IL-17 by CD4+CD25 T cells. ND, not detected.
Figure 5.
 
Adoptive transfer experiment to demonstrate the function of Treg cells in vivo. CD4+CD25+ Treg cells from control mice and immunized mice on days 14 and 28 were isolated and injected intravenously into naive syngeneic mice on the day of immunization with IRBP161–180 to induce EAU. The intensity of EAU on day 14 was scored in a masked fashion from 0 to 4 according to histopathologic grading. Transfer of CD4+CD25+ Treg cells from immunized mice obtained on day 14 or 28 could suppress EAU. Four mice were used in each group.
Figure 5.
 
Adoptive transfer experiment to demonstrate the function of Treg cells in vivo. CD4+CD25+ Treg cells from control mice and immunized mice on days 14 and 28 were isolated and injected intravenously into naive syngeneic mice on the day of immunization with IRBP161–180 to induce EAU. The intensity of EAU on day 14 was scored in a masked fashion from 0 to 4 according to histopathologic grading. Transfer of CD4+CD25+ Treg cells from immunized mice obtained on day 14 or 28 could suppress EAU. Four mice were used in each group.
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