J Korean Endocr Soc.  2007 Oct;22(5):332-338. 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

• Kim ES
• Jung HW
• Choi JI
• Nam-Goong
• Hong SH
• Kim YI

• KMID: 1523102
• DOI: http://doi.org/10.3803/jkes.2007.22.5.332

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.

Keyword

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

MeSH Terms

Autoantigens
Autoimmune Diseases
Coculture Techniques
Graves Disease*
Humans
Lymphocytes
Peripheral Tolerance
Receptors, Tumor Necrosis Factor
RNA, Messenger
T-Lymphocytes*
T-Lymphocytes, Regulatory*
Thyroid Diseases
Thyroid Gland
Autoantigens
RNA, Messenger
Receptors, Tumor Necrosis Factor

Figure

• 

  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).

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