ICR male mice (4 to 5 week old) were infected with HSV type 1 (1x106pfu/mL, F strain) grown in Vero cells, as previously described (Sohn, et al., 1998). Virus inoculation was performed twice with 10 day intervals, followed by 16 weeks of observation. Animals were handled in accordance with a protocol approved by the animal care committee of Ajou University School of Medicine.
disappearance of symptoms or more than 20% decrease in the lesion size were classified as effective. The severity score of BD was followed by determination of the value of Behcet’s disease activity index, as outlined in the BD Activity Form (www.behcet.ws/pdf/BehcetsDiseaseActivityForm.pdf). Among the symptoms in human patients, mouth ulceration, genital ulceration, erythema, skin pustules, skin ulceration, joints-arthritis, diarrhea, red eye (right, left), reduced vision (right, left), loss of balance, discoloration, and swelling of the face were selected and analyzed in the BD-like mouse models. The score of each symptom is one and after the score was added up, the total was used in determining the severity score of BD.
D. Flow cytometry
Mouse anti-CD4 and anti-CD25 antibodies are surface markers that were stained with samples for 30min at 4°C in the dark. For the intracellular detection of Foxp3, anti-mouse Foxp3 staining buffer set was used according to the manufacturer's instructions. Briefly, cells were fixed using Fix/perm buffer (according to the manufacture manual) after washing with 1X permeabilization buffer, then incubated with anti-mouse Foxp3 Ab for 30 min at 4°C in the dark. Stained cells were analyzed on a flow cytometer (FACS Vantage; Becton Dickinson) with ≥10,000 gated lymphocytes. A negative isotype control was used to distinguish stained from unstained cells.
E. Splenocytes culture and generation of Treg cells
The mice used in our experiments were 4 to 5 week old ICR male mice. Splenocytes
were isolated from mice and involved erythrocytes that were lysed by exposure to ACK solution. The cells were washed twice in a phosphate buffer saline (PBS). The splenocytes were in RPMI 1640 medium, supplemented 10% heat inactivate FBS and 1% Antibiotics, stimulated with anti-CD3 (0.1 ug/ml), anti-CD28 (0.2 ug/ml), rIL-2 (20 U/ml), and TGF-β1 (2 ng/ml). Then the cells were seeded at a density of 1.5 x 106cells per 6-well tissue culture plates and incubated for 1 to 4 days.
F. Dendritic cells isolation and culture
Dendritic cells (DCs) were derived from bone marrow cells flushed from tibias with a 26-gauge needle containing RPMI-1640 medium with 10% FBS and 1% antibiotics. Cells were dissociated through a 100-μm nylon mesh cell strainer and cultured in RPMI containing 15 ng/ml of GM-CSF. After 3 days of culture, floating and loosely adherent cells were collected and recultured in fresh RPMI-1640 supplemented with GM-CSF for an additional 7 days of culture. The DCs were then used within an additional 14 days.
Removal of adherent DCs was achieved by a 15min incubation (4°C) with 5 mM EDTA followed by gentle-trituration. Maturation of DCs was achieved by incubation with 500 ng/ml LPS for 24 h.
G. Co-culture of CD4+T cell with DCs
(anti-CD3 Ab, anti-CD28 Ab, rIL-2 and TGF-β). mDCs of 1x105cells and CD4+CD25+T of 1 x 106 cells in 12 well culture plates cultured for 72 h. After co-culture, harvest of suspension cells were stained with CD4, CD25, and Foxp3 and analyzed by flow cytometry.
H. Isolation of CD4+CD25+T cells
The splenocytes from normal mice were cultured for 2 days with stimulators.
CD4+CD25+T or CD4+CD25-T cells were isolated from cultured splenocytes by using a mouse CD4+CD25+ regulatory T cell isolation kit™ (Milteny Biotec, Auburn, CA), according to the manufacturer's instructions. Briefly, CD4+T cells were first isolated through negative selection by removing all other cell types. Preisolated CD4+T cells were incubated with magnetic beads conjugated with anti-CD25 antibody to separate CD4+CD25+T and CD4+CD25-T cell populations. Isolated T cells were >80% pure upon re-analysis by flow cytometry.
I. Adoptive transfer of CD4+CD25+T and CD4+CD25-T cells to BD-like mice
CD4+CD25+T and CD4+CD25-T cells isolated from normal splenocytes were sorted.
CD4+CD25+T cells were adoptively transferred to BD-like mice via a tail vein. The transferred cells were 3x103, 3x104, and 3x105 CD4+CD25+T cells (per mouse) which were cultured for 2 days from normal splenocytes. We observed the improvement of
symptoms by photographs 1 to 2 weeks after adoptive transfer. Two weeks later, BD-like mice transferred with CD4+CD25+T cells were sacrificed by cervical dislocation, and then serum and spleen tissues were collected.
J. ELISA
Two weeks after transferred with CD4+CD25+T cells to BD-like mice, the serum was obtained. Serum was analyzed using commercial ELISA kits for the detection of mouse IL-6, TNF-, TGF-β, IL-17, IFN-γ, and IL-10. All ELISA kits were purchased from R&D
systems (Minneapolis, MN). The ELISA was carried out according to the manufacturer’s instructions. Means and standard deviations were calculated using ELISA values determined for each well. The ELISA reader was a Bio-Rad model 170-6850 microplate reader, and wavelength was 450 nm.
K. Reverse transcription PCR (RT-PCR)
Total RNA was isolated with TRIzol (Life Technologies, Helgerman, CT), according to the manufacturer’s instructions. An amount of 1 g of total RNA was used as a template for cDNA synthesis with SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen, Carlsbad, CA). The cDNA was amplified by RT-PCR with the primers (Table 1). Amplified PCR products were visualized on 1.8% agarose gels.
Table 1. The sequence of primers used for RT-PCR.
* RORγt (Kimura, et al., 2007), TNF-α (Murray, et al., 1990), TGFβ (Derynck, et al., 1986), IL-17A (Hsu, et al., 2008), IL-17F(Yamaguchi, et al., 2007)
Gene sequence
L. Statistical analysis
All data are represented as the mean SE. Statistical differences between the experimental groups were determined using the Chi-square test, Student’s t test, and Bonferroni correction. Statistical analysis was performed using the MedCalc® version 9.3.0.0.
Ⅲ. RESULTS
A. CD4+CD25+ Treg cells in BD-like mice compared to BDN mice
We examined the frequencies of CD4+CD25+T and Treg cells in splenocytes of normal healthy, BDN (BD asymptomatic, HSV was inoculated but no symptomatic mice), and BD-like mice by FACS analysis. The CD4+CD25+T cell percentages in BD-like mice (n=9) were significantly lower than BDN mice (n=9)(0.59 ± 0.42 vs 1.14 ± 0.4%, p=0.012) and normal mice (n=8)(0.59 ± 0.42 vs 2.52 ± 1.31%, p=0.0008). Also BDN mice were lower than normal mice (1.14 ± 0.4 vs 2.52% ± 1.31%, p=0.009)(Fig 1A). Treg cell frequency in BD-like mice were lower than BDN mice (0.39 ± 0.37 vs 0.78 ± 0.56%, p=0.098) and normal mice (0.39 ± 0.37 vs1.15 ± 0.83%, p=0.028)(Fig 1B). CD4+CD25+
T and Treg cells in BDN mice were 2 times greater than BD-like mice. BD-like mice showed a decline in the frequency of Treg cells.
A B
Fig. 1. The frequencies of CD4+CD25+T and Treg cells in splenocytes of BDN and BD-like mice was compared by FACS analysis. CD4+CD25+T and Treg cell levels were lower in BD-like mice compared to BDN mice (A and B). A. The percentage of CD4+CD25+T cells was 0.59 ± 0.42% for BD-like mice (n=9) and 1.14 ± 0.4% for BDN mice (n=9)(BD-like vs BDN, p=0.012) and 2.52 ± 1.31% for normal mice (n=8)(BD-like vs normal, p=0.0008). B. The percentage of Treg cell was 0.39 ± 0.37% for BD-like mice and 0.78 ± 0.56% for BDN mice (BD-like vs BDN, p=0.098) and 1.15 ± 0.83% for normal mice (BD-like vs normal, p=0.0028). The short bars indicate the means. BD: Behcet’s Disease-like mice, BDN: BD Normal (BD asymptomatic) mice, CD4+CD25+: CD4+CD25+T cells, Treg: regulatory T (CD4+CD25+Foxp3+) cells
B. CD4+CD25+T cells were amplified in primary cultures of spleen tissues
Splenocytes were cultured with stimulators for several days, and then CD4+CD25+T cells and Treg cells were analyzed by FACS analysis. On day 0, Fig. 2 (A and B) show the frequencies of CD4+CD25+T cells of BD-like mice were 0.59±0.42% compared to 2.52±1.31% (p=0.0008) in normal mice, 1.14±0.4% (p=0.012) in BDN mice, and Treg cells of BD-like mice were 0.41±0.35% compared to 1.15±0.83% (p=0.028) in normal mice, 0.79±0.56% (p=0.098) in BDN mice. On day 1, the percentage of CD4+CD25+T cells of BD-like mice was 4.05±0.29% compared to 16.99±3.04% (p<0.01) in normal mice, 12.02±2.34% (p<0.01) in BDN mice and Treg cells of BD-like mice were 0.64±0.59% compared to 3.34±0.85% (p<0.01) in normal mice, 1.75±1.57% in BDN mice.The percentage of Treg cell in BDN was 2.4 times higher than BD-like mice, though statistically not significant. On day 2, the percentage of CD4+CD25+T cells of BD-like mice was 4.67±1.39% compared to 22.68±5.96% (p<0.01) in normal mice and 16.80±5.66% (p<0.05) in BDN mice. The frequency of Treg cell of BD-like mice was 0.74±0.46% compared to 3.37±1.86% (p<0.01) in normal mice and 1.75±0.94% in BDN mice. Treg cell in BDN was 2.3 times greater than BD-like mice though statistically not significant. On days 3 and 4, CD4+CD25+T and Treg cells of BD-like mice were lower than BDN or normal and resulted in a decreasing tendency.On day 3, the percentage of CD4+CD25+T cells of BD mice was 3.41±1.11% compared to 8.92±2.56% in the normal mice (p<0.05), 12.78±2.78% in the BDN mice (p<0.01) and Treg cell of BD-like mice was 0.85±0.74% compared to 1.35±0.84% in the normal mice, 1.18±1.02% in the BDN mice. On day 4, the percentage of CD4+CD25+ T cells of BD mice was 1.29±0.72%
compared to 6.20±1.71% in the normal mice (p<0.01), 4.55±0.39% in the BDN mice (p<0.01) and Treg cell of BD-like mice was 0.04±0.08% compared to 0.65±0.14% in the normal mice (p<0.01), 0.78±0.43% in the BDN mice (p<0.1). Our results show that CD4+CD25+ T and Treg cells of splenocytes in BD-like mice were lower than BDN or normal mice.
To confirm the proliferation of CD4+CD25+T and Treg cells, fold increase in proliferation after culture with stimulators was traced. CD4+CD25+T cells from normal mice increased 6±1, 8±2.1, 3±1.3, and 3±0.2 fold on day 1, 2, 3, and 4, respectively. BDN mice increased 11±2.9, 15±4.9, 11±3.4, and 4±0.5 fold on day 1, 2, 3, and 4, respectively.
BD-like mice increased 7±0.7, 8±3.3, 6±2.6, and 2±1.6 fold on day 1, 2, 3, and 4, respectively. (No cultured groups were standard and 1 fold). Treg cells in normal mice increased 3±0.7, 2.7±1.4, 1±0.9, and 0.6±0.1 fold on day 1, 2, 3, and 4, respectively. BDN mice increased 2.2±2.8, 2.2±1.2, 1.5±1.8, and 1±0.8 fold on day 1, 2, 3, and 4, respectively. BD-like mice increased 2.2±2.8, 2.2±1.2, 1.5±1.8, and 1±0.8 fold on day 1, 2, 3, and 4, respectively. The proliferation fold was not statistically significant among groups in normal, BDN, and BD-like mice.
The frequencies of CD4+CD25+T and Treg cell cultured for two days showed the highest expression compared to the other days. Therefore we used two day cultured splenocytes.
A B
Fig. 2. CD4+CD25+T cells were amplified in primary cultures of spleen tissues and amplified range was different among normal, BDN, and BD-like mice. The frequencies of CD4+CD25+T and Treg cells of splenocytes from normal, BDN, and BD-like mice were serially measured with stimulators at 0 (no culture), 1, 2, 3 and 4 day. CD4+CD25+T and Treg cells of BDN mice were compared to BD-like mice. The frequency of CD4+CD25+T cells in normal mice was significantly higher than BDN and BD-like mice. The frequency of CD4+CD25+T cells in BDN was significantly higher than BD-like mice (A). In Treg cells,
normal mice were significantly higher than BDN and BD-like mice at day 1 and 2 but the difference between BDN and BD-like mice was not significant on day 3 or 4(B). C and D:
The fold increase in proliferation CD4+CD25+T and Treg cell after culture with stimulators was traced. On day 2, CD4+CD25+T cells in BD-like mice were lower than the BDN group but statistically were not significant. In Treg cells, BD-like mice were lower than normal and BDN groups. Data are means ± SD of three independent experiments. *p<0.1, **p<0.05,
***p<0.01, NC : no culture (day 0), Nor: normal mice, BDN: BD asymptomatic mice, BD:
Behcet’s Disease-like mice, CD4+CD25+: CD4+CD25+T cells, Tregs: regulatory T (CD4+CD25+Foxp3+) cell
C. CD4+T cells from normal, BDN, and BD-like splenocytes were co-cultured with dendritic cells from normal mice.
To find the most important factor for proliferating CD4+CD25+T and Treg cells between CD4+T cells and dendritic cells (DC), CD4+T cells from normal, BDN, and BD splenocytes were co-cultured with dendritic cells from normal mice and DC from normal, BDN, and BD-like splenocytes were co-cultured with CD4+T cells from normal mice.
This study showed proliferation of CD4+CD25+T and Treg cells when CD4+T cells were co-cultured with antigen presenting DC for several days. DC from bone marrow of normal mice was isolated and cultured for 10 days. After LPS treatment, mature DCs (mDCs) were used. CD4+T cells were isolated from spleen tissues of normal, BDN, and BD-like mice. 1x105 mDCs from normal mice and 1 x 106 CD4+T cells from normal, BDN, and BD-like mice were co-cultured with anti-CD3 Ab, anti-CD28 Ab, rIL-2, and TGF-β for 72 h in a 12 well culture plate. Fig. 3A shows the frequency of CD4+CD25+T cells from normal mice (65%) was higher than BDN (47%) and BD-like (40%) mice. The Treg cells from normal mice (24%) were higher than BDN (10%) and BD-like (4%) mice.
CD4+CD25+T cell proliferation was affected by mDCs, but differences among groups were not significantly different. An important factor for the difference of proliferation of CD4+CD25+T cells among groups was the state of CD4+T cells. CD4+T cells were isolated from spleen tissues of normal mice. DCs were derived from normal, BDN, and BD-like mice. 1 x 106 CD4+T cells from normal mice and 1x105 mDCs from normal, BDN, and BD-like mice were co-cultured with anti-CD3 Ab, anti-CD28 Ab, rIL-2, and TGFβ for 72 h in a 12 well culture plate. Fig. 3B shows the expression of CD4+CD25+T
cells from normal DCs was 37%, 38% from BDN DCs, and 41% from BD-like mice DCs.
The percentage of CD4+CD25+T cells was a little different among the groups, but statistically not significant.
A
Fig. 3. CD4+T cells from normal, BDN, and BD-like splenocytes were co-cultured with dendritic cells from normal mice and DC from normal, BDN, and BD-like splenocytes were co-cultured with CD4+T cells from normal mice. Dendritic cells (DCs) from bone marrow of normal mice was isolated, cultured, and maturated. CD4+T cells were isolated from spleen tissues of normal, BDN, and BD-like mice. 1x105mDCs from normal mice and 1 x 106 CD4+T cells from normal, BDN, and BD-like mice were co-cultured with stimulators. Fig. 3A shows that frequency of CD4+CD25+T cells from normal mice was higher than BDN and BD-like mice. The Treg cells from BDN mice were higher than BD-like mice. B. CD4+T cells were isolated from spleen tissues of normal mice. DCs were derived from normal, BDN, and BD-like mice. 1 x 106 CD4+T cells from normal mice and 1x105 mDCs from normal, BDN, and BD-like mice were co-cultured with stimulators. The difference of CD4+CD25+T and Treg cells among the groups did not occurred. CD4+T cells are a more important factor than mDC in differenting the expression of CD4+CD25+T and Treg cells in normal, BDN, and BD-like mice. Nor mDC: normal mature dendritic cells, Nor CD4+ T cell: normal CD4+T cells
D. Adoptive transfer of CD4+CD25+T cells up-regulated the frequencies of Treg cell in BD-like mice
The CD4+CD25+T cells from splenocytes in normal healthy mice were cultured with stimulators and then isolated by MACS. After sorting, ≥80% of CD4+CD25+T cells was present in sorted fraction and CD4+CD25-T cells were ≥85% by FACS analysis (Fig. 4).
The proportions of Foxp3 positive cells were ≥25% in the CD4+CD25+T cells and ≤ 3%
in the CD4+CD25-T cells (Fig. 4). CD4+CD25+T and CD4+CD25-T cells were applied to in vivo BD-like mice. CD4+CD25+ (3 x 103, 3 x 104, and 3 x 105) T cells and CD4+CD25- (3 x 105) T cells were adoptively transferred by intravenous injection to BD-like mice. Two weeks after transfer with CD4+CD25+T cells, the transferred mice were sacrificed and the change of frequency of Treg cells from spleen tissues was identified.
The frequency of CD4+CD25+T cells after transfer with CD4+CD25+T cells is as follows; 0.59±0.42% for the not transferred group (n=9), 0.50±0.04% for the 3 x 103 group (n=3), 0.55±0.16% for the 3 x 104 group (n=5), and 0.95±0.39% for the 3 x 105 group (n=16) (not transfer vs 3 x 105, p=0.004; 3 x 103vs 3 x 105, p=0.04; 3 x 104vs 3 x 105, p=0.06)(Fig. 5A). The percentage of Treg cells transferred with CD4+CD25+T cells is as follows; 0.41±0.35% for the not transferred group, 0.31±0.01% for the 3 x 103group, 0.26±0.14% for the 3 x 104group, and 0.69±0.30% for the 3 x 105group (no transfer vs 3 x 105, p=0.04; 3 x 103 vs 3 x 105, p=0.04; 3 x 104 vs 3 x 105, p=0.004)(Fig. 5B). Our results show that transfer with 3 x 105 of CD4+CD25+T cells were significantly higher than 3 x 103and 3 x 104 of CD4+CD25+T cells. After transfer of CD4+CD25+T cells to BD-like mice, Treg cells were up-regulated.
uncultured
Fig. 4. CD4+CD25+T cells cultured and proliferated from normal splenocytes were isolated by MACS. The splenocytes of normal healthy mice were cultured for 2 days with stimulators (anti-CD3 Ab, anti-CD28 Ab, rIL-2, and TGF-β). Then, CD4+CD25+T or CD4+CD25-T cells were isolated by using a mouse CD4+CD25+ regulatory T cell isolation kit™ (Milteny Biotec, Auburn, CA) according to the manufacturer's instructions. After sorting by MACS, we checked CD4+CD25+T cells by FACS analysis (>80%). Also the proportions of Foxp3 positive cells were identified in the sorted CD4+CD25+T cells (≥25%).
A
No transfer 3x103 3x104 3x105 CD4+CD25+
No transfer 3x103 3x104 3x105 CD4+CD25+
No transfer 3x103 3x104 3x105 CD4+CD25+
No transfer 3x103 3x104 3x105 CD4+CD25+
p=0.04 p=0.04
p=0.004
Fig. 5. The frequencies of CD4+CD25+T and Treg cells in the CD4+CD25+T cells transferred BD-like mice. Various amounts of CD4+CD25+T cells were transferred via tail vein injection to like mice. Two weeks after transfer with CD4+CD25+T cells to BD-like mice, the mice were sacrificed, and the obtained spleen tissues containing CD4+CD25+T and Treg cells were analyzed by FACS. The frequencies CD4+CD25+T and
Treg cells in transferred BD-like mice were dependent on the amount of transferred cells.
The more CD4+CD25+T cells transferred and the more CD4+CD25+T and Treg cells remained significantly in the BD-like mice. CD4+CD25+: CD4+CD25+T cells, Treg:
regulatory T (CD4+CD25+Foxp3+) cells, no transfer: n=9, 3 x 103: n=3, 3 x 104: n=5, 3 x 105: n=18
E. Change of symptoms in BD-like mice after adoptive transfer
After injection of isolated CD4+CD25+T cells intravenously, CD4+CD25+T cell numbers increased by FACS analysis. Before and 2 weeks after transfer, the changes of symptoms in BD-like were photographed. The BD-like symptoms, such as oral ulcer, skin ulcer, scrotum enlargement, genital inflammation, arthritis, etc, were improved (Fig. 6). 3 x 105 of CD4+CD25+T cells effectively decreased BD-like symptoms in 15 of 18 cases (83%). But transfer with CD4+CD25-T cells decreased BD-like symptoms in 4 of 9 cases (33%) and 3 x 104 of CD4+CD25+T cells decreased BD-like symptoms in 4 of 7 cases (57%)(Table 2). CD4+CD25+T cells transfer brought the improvement of BD-like symptoms.
3 x 103 CD4+CD25+
CD4+CD25-Fig. 6. CD4+CD25+T or CD4+CD25-T cells were transferred to BD-like mice when their BD-like symptoms occurred. Photographs of mice were taken before and after transfer with CD4+CD25+T and CD4+CD25-T cells in BD-like mice. Transfer with 3 x 104 and 3 x 105 CD4+CD25+T cells to BD-like mice improved the BD-like symptoms but transfer with 3 x 103 CD4+CD25+T and CD4+CD25-T cells were not improved or deteriorated the BD-like symptoms. 3 x 105 CD4+CD25+T cells transferred BD-like mice were most effective in the improvement of symptoms.
Table 2. The change of symptoms after CD4+CD25+T cell transfer in BD-like mice
Change of symptoms Improved
No. /Total No. (%)
Dead /Total
(%) Improvement Deterioration
3 x 103 2/5 (40) 2/5 (40) Skin Eye, skin
3 x 104 4/7 (57) 2/7 (29) Skin Eye
CD4+CD25+
3 x 105 15/18 (83) 2/18 (11) Eye, genital, skin Skin
CD4+CD25- 4/9 (44) 2/9 (22) Skin Genital, Eye, skin Improvement and deterioration was decided by severity score.
F. The change of severity score in transferred with CD4+CD25+T cells to BD-like mice
The changes of symptoms are shown in Fig. 7. The BD severity score of adoptive transfer with 3 x 104 and 3 x 105 CD4+CD25+T cells in BD-like mice decreased statistically significant compared to adoptive transfer with CD4+CD25-T cells or 3 x 103 CD4+CD25+T cells in BD-like mice. The severity score of adoptive transfer with 3 x 103 CD4+CD25+T cells in BD-like mice was 2.71.2 before the injection and 1.70.6 after 2
weeks (p=0.225, n=3), 3 x 104CD4+CD25+T cells was 2.80.8 before and 1.81.3 after (p=0034, n=5), 3 x 105 CD4+CD25+T cells was 2.80.9 before and 1.30.9 after (p=0.0001, n=16), and CD4+CD25-T cells was 2.10.4 before and 1.71.0 after (p=0.143, n=9). Symptoms of transferred with CD4+CD25+T cells in BD-like mice were improved, but CD4+CD25-T cell transfer was not effective by improved.
0
Fig. 7. The comparison of the severity score before and after transfer of CD4+CD25+T or CD4+CD25-T cells in BD-like mice. Various amounts of CD4+CD25+T and CD4+CD25-T cells were adoptively transferred via tail vein injection to BD-like mice. The comparison of the disease severity score was calculated before and after transfer with CD4+CD25+T or CD4+CD25-T cells. The severity score was decreased and statistically significant in transferred with 3 x 104and 3 x 105CD4+CD25+T cells in BD-like mice.
G. CD4+CD25+T cells transfer up-regulated serum IL-10 and TGF-β levels and down-regulated IFN-γ and TNF- levels
Many studies have investigated the secretion of TGF-β and IL-10 by CD4+CD25+T cells (Dieckmann, et al., 2002; Papiernik, et al., 1997; Stephens, et al., 2001). It is known that the roles of IL-10 and TGF-ß in the suppressive effects are mediated by CD4+CD25+T cells. By ELISA, the IL-10 protein level was 75.3±48.5 pg/ml in the serum
Many studies have investigated the secretion of TGF-β and IL-10 by CD4+CD25+T cells (Dieckmann, et al., 2002; Papiernik, et al., 1997; Stephens, et al., 2001). It is known that the roles of IL-10 and TGF-ß in the suppressive effects are mediated by CD4+CD25+T cells. By ELISA, the IL-10 protein level was 75.3±48.5 pg/ml in the serum