Suppression of Pathogenic Autoreactive CD4+ T Cells by CD137-mediated Expansion of CD4+CD25+ Regulatory T Cells in Graves' Disease

Eun Sook Kim; Hyo Won Jung; Jung Il Choi; Il Seung Nam-Goong; Soon Hyung Hong; Young IL Kim

doi:10.3803/jkes.2007.22.5.332

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Kim, Jung, Choi, Nam-Goong, Hong, and Kim: Suppression of Pathogenic Autoreactive CD4+ T Cells by CD137-mediated Expansion of CD4+CD25+ Regulatory T Cells in Graves' Disease

Original Article

Journal of Korean Endocrine Society

Journal of Korean Endocrine Society 2007; 22(5): 332-338.

Published online: 20 October 2007

DOI: https://doi.org/10.3803/jkes.2007.22.5.332

Suppression of Pathogenic Autoreactive CD4⁺ T Cells by CD137-mediated Expansion of CD4⁺CD25⁺ Regulatory T Cells in Graves' Disease

Eun Sook Kim, Hyo Won Jung¹, Jung Il Choi¹, Il Seung Nam-Goong, Soon Hyung Hong, Young IL Kim

Department of Internal Medicine, Ulsan University Hospital, Ulsan University of Collage of Medicine, Korea.

¹Biomedical Research Center, Ulsan University Hospital, Ulsan University of Collage of Medicine, Korea.

Received 22 January 2007 Accepted 22 July 2007

Abstract

Background

Graves' disease (GD) is an organ-specific autoimmune disease that is characterized by lymphocyte infiltration of the thyroid, which finally leads to follicular destruction. The CD4^⁺CD25^⁺ regulatory T cells are important for maintaining peripheral tolerance to self-antigens and impaired activity can cause autoimmune diseases. CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily and expressed on activated T cells, is a candidate molecule for a co-stimulatory role in autoimmune thyroid disease. In this study, we aimed to assay the frequency of CD4⁺CD25⁺ T cells in GD patients and to investigate the role of CD137-mediated costimulation in CD4⁺CD25⁺ T cells.

Methods

The frequencies of the CD4⁺CD25⁺ T cells in the peripheral blood (PB) of GD patients were determined by flow cytometric analysis. After the CD4⁺CD25⁺ T cells were isolated from PB mononuclear cells (PBMC) of the GD patients using immunomagnetic beads, the functional activity of the CD4⁺CD25⁺ T cells was characterized by use of a proliferation assay. mRNA expression of Foxp 3 in the CD4⁺CD25⁺ T cells of the GD patients was observed by real-time RT-PCR.

Results

In this study, we found that GD patients had a low proportion of CD4⁺CD25⁺ T cells (mean ± SD; 1.47 ± 0.31%) in PBMC as compared with normal subjects. CD137-mediated costimulation increased the expression of CD25 and Foxp 3 in CD4⁺ T cells in GD patients as compared with normal subjects. Moreover, the CD137-mediated costimulation also induced the proliferation of CD4⁺CD25⁺ T cells in GD patients, and the expanded CD4⁺CD25⁺ T cells could suppress other CD4⁺CD25^- T cells in a co-culture.

Conclusion

These results suggest that the peripheral expansion of CD4⁺CD25⁺ T cells by CD137-mediated co-stimulation can suppress effector T cells and may be a potent therapy for Graves' disease.

Keywords: CD137-mediated costimulation, CD4⁺CD25⁺ T cells, Foxp 3, Graves' disease, Peripheral blood mononuclear cells

Figures and Tables

Fig. 1

The frequency of CD137⁺ cells in CD4⁺CD25⁺ T cells of the patients with Grave's disease. The frequency of CD25⁺CD4⁺ T cells in PBMC of GD patients and normal subjects were analyzed by flow cytometry with PE-labelled anti-CD137 staining. The values are expressed as mean ± SD of triplicate data. P < 0.0001 vs. normal group.

Fig. 2

The CD4⁺CD25⁺ T cell population in PBMC of patients with Graves' disease. PBMC were stimulated by plate-bound OKT3 (1 µg/mL), soluble anti-CD28 (1 µg/mL) and/or anti-CD137 (1 µg/mL) for 24 h. The cells in these analyses were gated on lymphocytes via forward and side scatter properties in flow cytometry. The values are expressed as mean ± SD of triplicate data. P < 0.05, P < 0.001 and P < 0.0001 vs. normal group.

Fig. 3

Proliferation of CD4⁺CD25⁺ T cells by anti-CD137 stimulation. The CD4⁺CD25⁺ T cells (5 × 10⁴ cells) were stimulated with APC (2 × 10⁴ cells/mL), plate-bound anti-CD3 (1 µg/mL), soluble anti-CD28 (1 µg/mL) and IL-2 (20 U/mL) at presence or absence with anti-CD137 mAb (4B4; 1 µg/mL). Proliferation was determined at day 3, with [³H]thymidine added for the last 18h of culture. The values are expressed as mean ± SD of triplicate data (SD = bar). P < 0.05 and P < 0.001 vs. normal group.

Fig. 4

The ability of the CD4⁺CD25⁺ T cells to suppress the proliferation of the cocultured CD4⁺CD25^- T cells. CD4⁺CD25⁺ T cells (2 × 10⁴ cells) were stimulated alone and in coculture with CD4⁺CD25^- T cells (2 × 10⁴ cells) in the presence of APC (2 × 10⁴ cells). The CD4⁺CD25^- T cells were also stimulated alone. Cells were stimulated with plate-bound anti-CD3(1 µg/mL) and soluble anti-CD28 (1 µg/mL) at presence or absence with anti-CD137 mAb (4B4; 1 µg/mL). Proliferation was determined at day 3, with [³H]thymidine added for the last 18h of culture.

Fig. 5

Expression of Foxp 3 in CD25⁺CD4⁺ T cells of GD patients. The CD4⁺CD25⁺ T cells were stimulated with OKT3/anti-CD28 (1 µg/mL) alone or with 4B4(1 µg/mL). After mRNA was isolated from cells using TRIzol reagent, cDNA was reverse transcribed and then amplified by PCR. Expression of Foxp 3 gene in CD4⁺CD25⁺ T cells of GD patients was determined by reverse transciption-PCR (A) and real time PCR (B).

Table 1

Clinical characteristics of GD patients and normal subjects

TBII, thyroid binding inhibitory immunoglobulin; TPOAb, antibodies to thyroid peroxidase; and TgAb, antibodies to thyroglobulin.

^†P < 0.001 for the comparison with normal control.

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