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단순포진바이러스로 유도된 베체트병 마우스모델에서 CD4+CD25+ 조절 T 세포의 역할

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The role of CD4+CD25+ regulatory T cells in

Herpes Simplex Virus-induced

Behcet's Disease-like mouse model

by

Ju A Shim

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

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The role of CD4+CD25+ regulatory T cells in

Herpes Simplex Virus-induced

Behcet's Disease-like mouse model

by

Ju A Shim

A Dissertation Submitted to The Graduate School of Ajou University

in Partial Fulfillment of the Requirements for the Degree of

Master of Biomedical Sciences

Supervised by

SeongHyang Sohn, Ph.D.

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

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This certifies that the dissertation

of Ju A Shim is approved.

SUPERVISORY COMMITTEE

SeongHyang Sohn

Eun-So Lee

Sun Park

The Graduate School, Ajou University

December, 22th, 2008

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ABSTRACT

-The role of CD4+CD25+ regulatory T cells in Herpes Simplex

Virus-induced Behcet’s Disease-like mouse model

Background: Behcet's disease (BD) is a chronic, multi-systemic disorder with arthritic,

gastrointestinal, mucocutaneous, ocular, vascular, and central nervous systems involved. CD4+CD25+ T regulatory (Treg) cells prevent harmful immune responses to self and non-self antigens. Treg cells play a crucial role in controlling and preventing autoimmunity. In this study, we investigated the role of Treg cells in BD-like mouse models.

Methods: Herpes simplex virus (HSV) type 1 inoculation of the earlobes of ICR mice

resulted in the development of BD-like symptoms. CD4+CD25+T cells in splenocytes of normal mice were adoptively transferred by intravenous injection to BD-like mice. We observed changes of pathologic manifestations during the 2 weeks after injection with CD4+CD25+T cells in BD-like mice. As a control, we transferred CD4+CD25-T cells to BD-like mice. Two weeks after injection, sera and splenocytes were collected. We analyzed TGF-β, IL-10, IFN-γ, TNF-α, IL-17, and IL-6 levels by ELISA or RT-PCR and also the frequency of Treg cells by FACS.

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compared to the transferred of 3x103and 3x104CD4+CD25+T cells. We also observed the

improvement of symptoms in BD-like mice after adoptive transfer with 3x105 of

CD4+CD25+T cells. And adoptive transfer with 3x103CD4+CD25+T cells or

CD4+CD25-T cells did not affect the symptoms of BD-like mice. Also, protein level IL-10 was

significantly increased in transferred with 3x105 CD4+CD25+T cells in BD-like mice

compared to not transferred BD-like mice (156.2 ± 80.1 vs 75.3 ± 48.5 pg/ml, p=0.03) or

transferred with 3x104 CD4+CD25+T cells to BD-like mice (156.2 ± 80.1 vs 54.8 ± 67.8

pg/ml, p=0.04). Protein levels of IL-6 and IL-17 were significantly decreased after transfer

with 3x105 CD4+CD25+T cells to BD-like mice compared to the not transferred BD-like

mice (IL-6: 39.2 ± 50.8 vs 215.6 ± 133.3 pg/ml, p=0.0002; IL-17: 19.1 ± 20.0 vs 38.0 ± 20.5 pg/ml, p=0.03). mRNA expression showed similar results with protein levels of TGF-β, TNF-α, IFN-γ, IL-6, and IL-17.

Conclusion: We confirmed the influence of CD4+CD25+T cells on BD-like mice. These

results show up-regulation of CD4+CD25+T cells to BD-like mice improved the

inflammatory symptoms and change of cytokine levels.We showed that CD4+CD25+T cells

alleviated the inflammation induced by autoimmune or autoinflammatory T cells.

Key words: Regulatory T cells, Herpes simplex virus-induced systemic inflammation,

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TABLE OF CONTENTS

ABSTRACT ··· ⅰ TABLE OF CONTENTS ··· ⅲ LIST OF FIGURE ··· ⅴ LIST OF TABLES ··· ⅶ Ⅰ. INTRODUCTION ··· 1

Ⅱ. MATERIALS AND METHODS ··· 4

A. Antibodies and reagents ··· 4

B. Animal experiments··· 4

C. BD-like symptoms ··· 4

D. Flow cytometry ··· 5

E. Splenocyte culture and generation of Treg cells ··· 5

F. Dendritic cells isolation and culture ··· 6

G. Co-culture of CD4+T cells with DCs ··· 6

H. Isolation of CD4+CD25+T cells ··· 7

I. Adoptive transfer of CD4+CD25+T and CD4+CD25-T cells to BD-like mice ··· 7

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A. CD4+CD25+ Treg cells in BD-like mice compared to BDN mice ··· 11

B. CD4+CD25+T cells were amplified in primary cultures of spleen tissues ··· 13

C. CD4+T cells from normal, BDN, and BD-like splenocytes were co-cultured with normal dendritic cells from normal mice ··· 17

D. Adoptive transfer with CD4+CD25+T cells up-regulated the frequencies of T cell in BD-like mice ··· 21

E. Change of symptoms in BD-like mice after adoptive transfer ··· 25

F. The change of severity score in transferred with CD4+CD25+T cells to BD-like mice ··· 28

G. CD4+CD25+T cells transfer up-regulated serum IL-10 and TGF-β levels and down-regulated IFN-γ and TNF-α levels ··· 31

H. Serum IL-6 and IL-17 levels were down-regulated after transfer ··· 34

I. The differences of serum cytokine amounts between the average of not transferred BD-like mice and 3x105CD4+CD25+T cells transferred each BD-like mouse ··· 39

Ⅳ. DISCUSSION ··· 45

Ⅴ. CONCLUSION ··· 49

REFERENCES ··· 50

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LIST OF FIGURES

Fig. 1. The frequencies of CD4+CD25+T and Treg cells in splenocytes of BDN

and BD-like mice was compared by FACS analysis ··· 12

Fig. 2. CD4+CD25+T cells were amplified in primary cultures of

splenocytes and amplified range was different among normal, BDN,

and BD-like mice ··· 15

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.··· 19

Fig. 4. CD4+CD25+T cells cultured and proliferated from normal splenocytes were isolated by MACS ··· 22

Fig. 5. The frequencies of CD4+CD25+T and Treg cell in the

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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 ··· 30

Fig. 8. Protein and mRNA levels of cytokines were compared between BD-like mice and CD4+CD25+T cells transferred BD-like mice ··· 36

Fig. 9. The differences of serum cytokine amounts between the average of

not transferred BD-like mice and 3x105CD4+CD25+T cell transferred each

BD-like mice by ELISA. The difference is displayed separately in BD skin symptomatic mice (BD skin) and BD ocular symptomatic mice with

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LISIT of TABLES

Table 1. The sequence of primers used for RT-PCR ··· 9

Table 2. The change of symptoms after CD4+CD25+T cell transfer in

BD-like mice ··· 28

Table 3. The serum cytokine level from each CD4+CD25+T cells transferred BD-like mice and the difference of between CD4+CD25+T cells

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Ⅰ. INTRODUCTION

Behcet’s Disease (BD) is a chronic, multi-systemic disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular, and central nervous system involved. This disease takes a chronic course with periodic exacerbations and progressive deterioration (Zouboulis, et al., 2000; Choi, et al., 2008). The etiology of BD is unclear, but viral infection has long been postulated as one of the main factors. Since Hulusi Behcet first proposed a viral etiology (Seçkin, et al., 1995), the viral hypothesis has been verified by detection of virus in saliva (Müftüoğlu, et al., 1986), intestinal ulcers (al-Dalaan, et al., 1995), and genital ulcers (Katsantonis, et al., 2000; Sayinalp, et al., 1996) of patients with BD. Subsequent to these findings, herpes simplex virus (HSV) inoculation of the earlobes of ICR mice resulted in the development of BD-like symptoms (Sohn, et al., 1998). Manifestations in mice after HSV inoculation included multiple symptoms such as oral ulcers, genital ulcers, skin ulcers, eye symptoms, gastrointestinal ulcers, arthritis, and neural involvement, as well as skin crusting. The frequencies of these symptoms are similar to that of patients with BD (Adam, et al., 2004).

Sakaguchi identified a subpopulation of CD4+T cells constitutively expressing the IL-2 receptor α-chain (CD25) in 1995. CD4+CD25+ T regulatory (Treg) cells are heterogeneous T cell populations that prevent harmful immune responses to self and non-self antigens. Treg cells emerge from the thymus as part of normal T lymphocyte development and constitute

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5-10% of CD4+T cells in the periphery. Treg cells reside in peripheral tissues to maintain self-tolerance and to prevent autoimmunity by inhibiting pathogenic lymphocytes. Two groups of CD4+CD25+ Treg cells were found naturally occurring CD4+CD25+ Treg cells (nTreg) and inducible CD4+CD25+ Treg cells (iTreg) (Sakaguchi, 2005; Piccirillo, et al., 2004; Thompson, et al., 2004). nTreg cells, characterized by the expression of CD4, CD25, and the transcriptional factor Forkhead box P3 (forkhead / winged helix transcription factor; Foxp3), develop in the thymus and recognize specific self-antigen (Stephens, et al., 2007). Subset of Treg cells are also generated in the periphery during an active immune response and are called iTreg and the newly induced CD4+ suppressor cells inhibited proliferation of CD4+T cells either via interlukin (IL)-10 (Dieckmann, et al., 2001) or transforming growth factor (TGF)-β (Jonuleit, et al., 2002) production. Foxp3 protein is currently considered as the most specific marker of Treg cells and a mutation of this transcription factor is strongly linked to immune dysregulation. Treg cells play an important role in the pathogenesis of

autoimmune disorders, such as diabetes mellitus(Kukreja, et al., 2002), arthritis (Amelsfort,

et al., 2004 ; Cao D, 2003), and lupus (Crispin, et al., 2003). These autoimmune disorders

can be prevented by infusion of Treg cells (Sakaguchi, et al., 2001). More recently Yuki Nanke et al. reported that the percentage of Treg cells decreased in peripheral blood of patients with BD before ocular attack (Nanke, et al., 2008). In this study, we investigated the role of Treg cells by identifying and analyzing the function of CD4+CD25+ Treg cells in

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weeks after transferred with CD4+CD25+T cells, we analyzed frequencies of CD4+CD25+T and Treg cells in splenocytes and cytokines in sera and spleen tissues. The aim of this study was the affect on the inhibition of the progress of this severe inflammatory disease by elevating CD4+CD25+T cells in BD-like mice.

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Ⅱ. MATERALS AND METHODS

A. Antibodies and Reagents

Mouse anti-CD4 (FITC), anti-CD25 (PE), anti-CD3, anti-CD28 antibodies and recombinant mouse IL-2 (rIL-2) were purchased from BD Pharmingen (San Diego, CA). Mouse anti-Foxp3 (PE-cy5) antibody and Foxp3 staining buffer kit and granulocyte macrophage colony-stimulating factor (GM-CSF) were purchased from eBioscience (San Diego, CA). Recombinant human TGF-β1 was purchased from R&D system (Minneapolis, MN). RPMI-1640 and Fetal Bovine Serum (FBS) was purchased from Gibco, Invitrogen (Carlsbad, CA).

B. Animal experiments

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.

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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

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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 106

cells 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.

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(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

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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.

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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 F 5'-TGGAATCCTGTGGCATCCATGAAAC-3' β-actin R 5'-TAAAACGCAGCTCAGTAACA GTCCG-3' F 5'-TGGAGTCACAGA AGGAGTGGCTAAG-3' IL-6 R 5'-TCTGACCACAGTGAGGAATGTCCAC-3' F 5'- GCGGAGCAGACACACTTACA-3' RORγt R 3'-TTGGCAAACTCCACCACATA-5' F 5'-GGCAGGTCTACTTTGGAGTCATTGC-3' TNF-α R 5'-ACATTCGAGGCTCCAGTGAA TTCGG-3' F 5'-AGGAGACGGAATACAGGGCTTTCG-3' TGFβ R 5'-ATCCACTTCCAACCCAGGTCCTTC-3' F 5'-AGGCCCTCAGACTACCTCAACC-3' IL-17A R 5'- GCCTCTGAATCCACATTCCTTG-3' F 5'- CAACGCTGCATACAAAAATCA-3' IL-17F R 5'-TTAAGTGAGGCATTGGGAACA-3' F 5'-AGCGGCTGACTGAACTCAGATTGTAGCTTGTACCTTTACTTCACTG-3' IFN-γ R 5'-GTCACAGTTTTCAGCTGTATAGGG-3'

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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.

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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.

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A B CD4+CD25+ 0 2 4 6 % Nor BDN BD p=0.009 p=0.008 p=0.012 CD4+CD25+ 0 2 4 6 % Nor BDN BD p=0.009 p=0.008 p=0.012 Treg 0 1 2 3 4 % Nor BDN BD p=0.028 p=0.098 Treg 0 1 2 3 4 % Nor BDN BD p=0.028 p=0.098

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

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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%

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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.

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A B CD4+CD25+ 0 10 20 30

NC 1 day 2 day 3 day 4 day

% Nor BDN BD *** *** ** *** ** *** *** *** *** ** CD4+CD25+ 0 10 20 30

NC 1 day 2 day 3 day 4 day

% Nor BDN BD *** *** ** *** ** *** *** *** *** ** T regs 0 2 4 6

NC 1 day 2 day 3 day 4 day

% Nor BDN BD *** *** * *** * *** T regs 0 2 4 6

NC 1 day 2 day 3 day 4 day

% Nor BDN BD *** *** * *** * *** C D CD4+CD25+ 0 5 10 15 20 NC 1d 2d 3d 4d fo ld Nor BDN BD CD4+CD25+ 0 5 10 15 20 NC 1d 2d 3d 4d fo ld Nor BDN BD T reg 0 1 2 3 4 5 NC 1d 2d 3d 4d fo ld Nor BDN BD

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,

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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

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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

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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.

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A

CD4+ T cell

Nor BDN BD CD4 C D 2 5 40% 65% 47% CD25 F o x p 3 4% 24% 10% Nor mDC

CD4+ T cell

Nor BDN BD CD4 C D 2 5 40% 40% 65% 65% 47%47% CD25 F o x p 3 4% 4% 24% 24% 10%10% Nor mDC B

mDC

Nor BDN BD Nor CD4+ T cell C D 2 5 F o x p 3 CD4 CD25 37% 38% 41% 6 % 6 % 6 %

mDC

Nor BDN BD Nor CD4+ T cell C D 2 5 F o x p 3 CD4 CD25 37% 38% 41% 6 % 6 % 6 %

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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

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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

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uncultured CD4 C D 2 5 stimulated 1% 20% 80% 80% Foxp3 sorted 25% 3% CD4 C D 2 5 uncultured CD4 C D 2 5 stimulated 1% 20% 80% 80% Foxp3 sorted 25% 3% uncultured CD4 C D 2 5 stimulated 1% 20% 80% 80% Foxp3 sorted 25% 3% CD4 C D 2 5

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%).

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A CD4+CD25+ 0.0 0.6 1.2 1.8 % No transfer 3x103 3x104 3x105 CD4+CD25+ p=0.04 p=0.06 p=0.04 CD4+CD25+ 0.0 0.6 1.2 1.8 % No transfer 3x103 3x104 3x105 CD4+CD25+ p=0.04 p=0.06 p=0.04 B T regs 0.0 0.5 1.0 1.5 % No transfer 3x103 3x104 3x105 CD4+CD25+ p=0.04 p=0.04 p=0.004 T regs 0.0 0.5 1.0 1.5 % 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

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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

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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.

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3 x 103 CD4+CD25+ Before After 3 x 104 CD4+CD25+ 3 x 105 CD4+CD25+ No transfer Before 3 x 105 CD4+CD25-3 x 103 CD4+CD25+ Before After 3 x 104 CD4+CD25+ 3 x 105 CD4+CD25+ No transfer Before 3 x 103 CD4+CD25+ Before After 3 x 104 CD4+CD25+ 3 x 105 CD4+CD25+ No transfer Before 3 x 105

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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

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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

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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 2.71.2 before the injection and 1.70.6 after 2

weeks (p=0.225, n=3), 3 x 104CD4+CD25+T cells was 2.80.8 before and 1.81.3 after

(p=0034, n=5), 3 x 105 CD4+CD25+T cells was 2.80.9 before and 1.30.9 after

(p=0.0001, n=16), and CD4+CD25-T cells was 2.10.4 before and 1.71.0 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.

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0 1 2 3 4 before after 3 x 103 3 x 104 3 x 105 CD4+CD25+ CD4+CD25-p=0.225 p=0.143 p=0.034 p=0.0001 S e v e ri ty s c o re 0 1 2 3 4 before after 3 x 103 3 x 104 3 x 105 CD4+CD25+ CD4+CD25-p=0.225 p=0.143 p=0.034 p=0.0001 0 1 2 3 4 before after 3 x 103 3 x 104 3 x 105 CD4+CD25+ CD4+CD25-p=0.225 p=0.143 p=0.034 p=0.0001 S e v e ri ty s c o re

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

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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

of the not transferred BD-like mice (n=7) compared to 156.2±80.1 pg/ml in the 3 x 105

CD4+CD25+T cells transferred BD-like mice (n=13)(not transferred BD-like mice vs 3 x

105, p=0.03) and 54.8±67.8 pg/ml in the 3 x 104cells transferred BD-like mice (n=4)(3 x

104 vs 3 x 105, p=0.04), and 88.9±84.0 pg/ml in the BDN mice (n=7)(Fig. 8A). IL-10

level was increased after transfer with 3 x 104and 3 x 105CD4+CD25+T cells in the

BD-like mice. The importance of TGF-β in the immune system was highlighted by the discovery that TGF-β-deficient mice developed multiple inflammatory diseases (Kulkarni,

et al., 1993; Shull, et al., 1992). TGF-β expression was higher in transfer of 3 x 105 cells to BD-like mice than not transferred BD-like mice by RT-PCR and ELISA. By ELISA,

the TGF-β protein level was 19.8±20.0 pg/ml in the transfer with 3 x 105 CD4+CD25+T

cells in BD-like mice (n=15) compared to 4.7±1.9 pg/ml in the not transferred BD-like

mice (n=8), 10.4±7.3 pg/ml in the BDN mice (n=6), and 5.0±2.4pg/ml in the 3 x 104

CD4+CD25+ T cell transferred BD-like mice (n=5)(not transferred vs 3 x 105, p=0.08;

BD-like vs BDN, p=0.07)(Fig 8B). mRNA level of TGF-β in spleen tissues of transferred

with 3 x 105 CD4+CD25+T cells was higher than the not transferred BD-like (Fig 8G).

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levels were shown to be similar to BDN or higher than BDN. It was already known CD4+CD25+ Treg cells secrete IL-10 and TGF-β.

Interferon (IFN)-γ represents the key cytokine produced by Th1 cells. IFN-γ protein levels were higher in BD-like mice than BDN mice (Sohn, et al., 2001). IFN-γ levels were

compared between 3 x 105 CD4+CD25+T cells transferred BD-like mice and not

transferred BD-like mice. Our results indicated that transfer with 3 x 105 CD4+CD25+T

cells was significantly lower than not transferred BD-like mice by ELISA. IFN-γ levels

were 10.0±6.2 pg/ml for the transfer with 3 x 105 CD4+CD25+T cells in BD-like mice

(n=12) compared to 37.4±6.6 pg/ml in the not transferred BD-like mice (n=5), 5.3±7.4

pg/ml in the BDN mice (n=4), and 14.1±10.7 pg/ml in the transfer with 3 x 104

CD4+CD25+T cells BD-like mice (n=5)(not transferred vs 3 x 105, p=0.000001; BD-like

mice vs BDN, p=0.002; not transferred vs 3 x 104, p=0.03)(Fig. 8C). mRNA expression of

IFN- γ in spleen tissues of transferred with 3 x 105

CD4+CD25+T cells was lower than the not transferred BD-like mice (Fig 8G). Tumor necrosis factor- (TNF-) is a potent paracrine and endocrine mediator of inflammatory and immune functions. TNF- over expression has been implicated in acute and chronic inflammatory diseases, such as rheumatoid arthritis, atopic dermatitis, psoriasis, and Behcet’s disease (Akdeniz, et al.,

2004). TNF- levels were lower in 3 x 105

CD4+CD25+T cell transferred BD-like mice than not transferred BD-like mice by RT-PCR and ELISA. By ELISA, the TNF- level

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CD4+CD25+T cells to BD-like mice (n=5)(not transfer vs 3 x 105, p=0.002; BD-like mice

vs BDN, p=0.00005; 3 x 104 vs 3 x 105, p=0.002)(Fig 8D). mRNA expression of TNF-

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H. Serum IL-6 and IL-17 levels were down-regulated after transfer

It is known that IL-6, in combination with TGF-β, induces Th-17 cell generation from naïve T cells and inhibit TGF-β-induced Foxp3 expression (Kimura, et al., 2007; Fantini, et al., 2004). Th-17 cells produce IL-17A (IL-17) and IL-17F and, to a lesser extent, TNF and IL-6 (Langrish, et al., 2005). Th-17 cells are present in human patients with various autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematous, and asthma (Matusevicius, et al., 1999; Wong, et al., 2000; Hashimoto, et al., 2005; Lindén, et al., 2000). We confirmed that the serum IL-6 levels were higher in BD-like mice than BDN mice. Serum IL-6 levels were down-regulated by transfer of CD4+CD25+T cells in BD-like mice. The IL-6 protein level was 215.6±133.3 pg/ml for not transferred BD-like mice (n=6) compared to 39.2±50.8 pg/ml in the

transferred with 3 x 105 CD4+CD25+T cells in BD-like mice (n=16)(p=0.0002) and

75.8±60.9 pg/ml for the transferred with 3 x 104 CD4+CD25+T cells in BD-like mice

(n=5) (not transferred vs 3 x 105, p=0.06). In BDN mice (n=6), IL-6 levels were 19.9±20.3

pg/ml (BD-like vs BDN p=0.005)(Fig. 8E). So, all transferred mice were significantly lower than BD-like mice. Adoptive transfer of CD4+CD25+T cells could influence the expression of Th-17, thus, IL-17 protein production was measured by ELISA. The IL-17

protein level was 19.1±8.1 pg/ml in the transferred with 3 x 105 CD4+CD25+T cells in

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IL-17A and IL-17F in spleen tissues transferred with 3 x 105CD4+CD25+T cells and not transferred BD-like mice showed similar patterns to ELISA results. mRNA levels of

ROR-γt, transcription factor of Th17, was lower in 3 x 105

CD4+CD25+T cell transferred BD-like mice than the not transferred BD-like mice. From these, adoptive transfer of CD4+CD25+T cells altered the cytokine expression and improvement of BD-like symptoms.

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A B IL-10 0 100 200 300 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.03 p=0.04 IL-10 0 100 200 300 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ IL-10 0 100 200 300 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.03 p=0.04 TGF-beta 0 20 40 60 80 n g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.07 p=0.08 TGF-beta 0 20 40 60 80 n g /m l BDN BD 3x10 5 3x104 CD4+CD25+ TGF-beta 0 20 40 60 80 n g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.07 p=0.08 C D IFN-gamma 0 20 40 60 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.000001 p=0.002 p=0.03 IFN-gamma 0 20 40 60 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ IFN-gamma 0 20 40 60 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.000001 p=0.002 p=0.03 TNF-alpha 0 20 40 60 80 100 120 140 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.00005 p=0.002 p=0.002 TNF-alpha 0 20 40 60 80 100 120 140 p g /m l BDN BD 3x10 5 3x104 CD4+CD25+ p=0.00005 p=0.002 p=0.002 E F IL-6 200 300 400 p g /m l p=0.0002 p=0.06 p=0.005 IL-6 200 300 400 p g /m l IL-6 200 300 400 p g /m l p=0.0002 p=0.06 p=0.005 IL-17 40 60 80 100 p g /m l p=0.002 p=0.002 p=0.03 IL-17 40 60 80 100 p g /m l IL-17 40 60 80 100 p g /m l p=0.002 p=0.002 p=0.03

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G IL-6 TNF-α BD BDN 3x104 3x105 IL-17A IL-17F IFN-γ ROR- γt β-actin TGF-β CD4+CD25+ IL-6 TNF-α BD BDN 3x104 3x105 IL-17A IL-17F IFN-γ ROR- γt β-actin TGF-β CD4+CD25+

Fig. 8. Protein and mRNA levels of cytokines were compared between BD-like mice

and CD4+CD25+T cells transferred BD-like mice. Serum was collected from hearts of

mice at 2 weeks after transferred with CD4+CD25+T cells. Transfer with 3 x 104and 3 x 105

CD4+CD25+T cells in BD-like mice were compared to not transferred BD-like and BDN

mice. A~B: IL-10 and TGF-β protein levels were increased in transfer with 3 x 105

CD4+CD25+T cells to BD-like mice. C~F: IFN-γ, TNF-α, IL-6 and IL-17 protein levels

were decreased in transfer with 3 x 105CD4+CD25+T cells to BD-like mice. The short bars

indicate the means

.

G: mRNA expressions of cytokines in spleen tissues of BD-like mice,

BDN, and transferred with 3 x 104 and 3 x 105 CD4+CD25+T cells to BD-like mice,

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CD4+CD25+T cells to BD-like mice, Lane 4: transferred with 3 x 105 CD4+CD25+T cells to BD-like mice. mRNA levels of IFN-γ, TNF-α, IL-6, IL-17A, IL-17, and ROR-γt in

transferred with 3 x 105CD4+CD25+T cells were detected lower than not transferred

BD-like mice. But mRNA level of TGF-β in transferred with 3 x 105 CD4+CD25+T cells was

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I. The differences of serum cytokine amounts between the average of not

transferred BD-like mice and 3x10

5

CD4+CD25+T cells transferred each

BD-like mouse

The differences of serum cytokine amounts between the average of not transferred

BD-like mice and 3x105 CD4+CD25+T cell transferred each BD-like mouse were

displayed and BD skin symptomatic mice (BD skin) and BD ocular symptomatic mice with skin symptom (BD eye) displayed separately because it was reported that the immunology between BD skin and BD eye was different (Shon, 2007). The protein amount of CD4+CD25+T cells transferred BD-like mice by ELISA was subtracted from the not transferred average of BD-like mice (Fig 8). IL-10 protein levels were 78.9±67.4 pg/ml in BD skin (n=9) compared to 38.7±106.8 pg/ml in BD eye (n=7)(p=0.36), therefore, transferred mice up-regulated IL-10 protein levels and BD eye mice was lower than BD skin mice, but not significant. TGF-β protein levels were 11.1±16.5 ng/ml in BD skin (n=9) compared to 12.3±21.9 ng/ml in BD eye (n=7), so, transferred mice up-regulated TGF-β protein levels and the level of BD skin and BD eye mice showed similar results. IFN-γ protein levels were -23.0±10.5 pg/ml in BD skin (n=9) compared to -29.7±7.0 pg/ml in BD eye (n=7)(p=0.16), therefore, CD4+CD25+T cells transfer down-regulated IFN-γ and BD eye was lower than BD skin but not significant. TNFα protein levels were -26.8±9.6 pg/ml in BD skin (n=9) and -25.3±11.7 pg/ml in BD eye (n=7), so, TNF-α protein levels were decreased by CD4+CD25+T cells transfer but BD skin and BD eye were not different. IL-6 protein levels were -192.4±32.4 pg/ml in BD skin (n=9) compared to -155.8±64.6 pg/ml in BD eye (n=7)(p=0.16), thus, BD eye was higher than BD skin but

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not significant. IL17 protein levels were 23.2±4.1 pg/ml in BD skin (n=9) compared to -13.2±8.7 pg/ml in BD eye (n=7)(p=0.009), so, BD eye was significantly higher than BD skin. Interestingly, IL-17 only showed statistically significant difference between BD skin and BD eye. Also IFN-γ was affected more in BD eye than BD skin.

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A B IL-10 -100 -50 0 50 100 150 200 250 p=0.37 D if fe re n c e (p g /m l) skin eye IL-10 -100 -50 0 50 100 150 200 250 p=0.37 D if fe re n c e (p g /m l) skin eye TGF-beta -10 0 10 20 30 40 50 60 70 D if fe re n c e (n g /m l) skin eye TGF-beta -10 0 10 20 30 40 50 60 70 D if fe re n c e (n g /m l) skin eye C D IFN-gamma -50 -40 -30 -20 -10 0 p=0.16 D if fe re n c e (p g /m l) skin eye IFN-gamma -50 -40 -30 -20 -10 0 p=0.16 D if fe re n c e (p g /m l) skin eye p=0.16 D if fe re n c e (p g /m l) skin eye TNF-alpha -50 -40 -30 -20 -10 0 skin eye D if fe re n c e (p g /m l) TNF-alpha -50 -40 -30 -20 -10 0 skin eye D if fe re n c e (p g /m l) E F IL-6 -250 -200 -150 -100 -50 0 p=0.16 D if fe re n c e (p g /m l) skin eye IL-6 -250 -200 -150 -100 -50 0 p=0.16 D if fe re n c e (p g /m l) skin eye IL-17 -30 -20 -10 0 10 p=0.009 skin eye D if fe re n c e (p g /m l) IL-17 -30 -20 -10 0 10 p=0.009 skin eye D if fe re n c e (p g /m l)

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Fig. 9. The differences of serum cytokine amounts between the average of not

transferred BD-like mice and 3x105CD4+CD25+T cells transferred each BD-like mice

by ELISA. The difference is displayed separately in BD skin symptomatic mice (BD

skin) and BD ocular symptomatic mice with skin symptom (BD eye).

A. IL-10 protein level in BD skin was higher than BD eye, B. TGF-β protein level was not different in BD skin and BD eye, C. IFN-γ protein level in BD eye was lower than BD skin but not significant, D. TNF-α protein level was not different between BD skin and BD eye, E. IL-6 protein level in BD eye was higher than BD skin but not significant, F. IL-17 protein level in BD eye was significantly higher than BD skin. The short bars indicate the means. BD skin: BD skin symptomatic mice (n=9), BD eye: BD ocular symptomatic mice with skin symptom (n=7), Difference: The amount of protein by ELISA in CD4+CD25+T cell transferred BD-like mice subtracted by the average of not transferred BD-like mice

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Table 3. The serum cytokine level from each CD4+CD25+T cells transferred BD-like mice and the difference of between

CD4+CD25+T cells transferred BD-like mice and not transferred BD-like mice average.

IL-10 (pg/ml) TGF-β (ng/ml) IFN-γ (pg/ml) TNF-α (pg/ml) IL-6 (pg/ml) IL-17 (pg/ml) Mouse

No. Transfer Difference Transfer Difference Transfer Difference Transfer Difference Transfer Difference Transfer Difference

1 195.6 120.3 10.5 5.9 6.3 -31.1 47.4 -20.4 60.2 -155.0 15.8 -22.2 2 117 41.7 4.7 0.0 12.7 -24.7 32.4 -35.4 37.1 -179.0 13.8 -24.2 3 219.1 143.8 4.2 -0.5 28.7 -8.7 39.5 -28.3 0.0 -216.0 10.0 -28.0 4 229.3 154.0 11.1 6.4 2.6 -34.8 36.8 -31.0 0.0 -216.0 16.4 -21.6 5 179.3 104.0 11.8 7.1 31.3 -6.1 54.4 -13.4 19.2 -196.0 17.8 -20.2 6 86.3 11.0 54.7 50.0 22.0 -15.4 40.6 -27.2 0.0 -216.0 12.1 -25.9 7 206.5 131.5 29.5 24.8 6.3 -31.1 55.0 -12.8 88.7 -127.0 21.6 -16.4 8 35.8 -40.0 3.4 -1.3 12.9 -24.5 35.7 -32.1 0.0 -216.0 19.3 -18.7 9 119.0 43.7 12.7 8.0 8.4 -29.0 27.1 -40.7 3.6 -212.0 11.8 -26.2 10 236.4 161.1 65.8 61.1 7.9 -29.5 36.8 -31.0 167.5 -48.1 26.8 -11.2 11 120.0 44.7 15.6 10.9 0.2 -37.3 37.9 -30.0 127.1 -88.5 26.6 -11.4 12 77.1 1.8 9.0 4.3 10.2 -27.2 63.3 -4.5 69.3 -146.0 22.8 -15.2 13 57.7 -17.6 7.8 3.1 20.0 -17.4 30.8 -37.0 3.82 -212.0 21.6 -16.4 14 25.0 -50.3 12.5 7.8 1.8 -35.7 53.8 -14.0 26.9 -189.0 42.3 4.3

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15 0.0 -75.3 4.8 0.1 2.4 -35.0 34.0 -33.8 7.92 -208.0 17.0 -21.0

16 281.4 206.0 3.5 -1.2 11.4 -26.0 41.2 -26.6 16.4 -199.0 10.6 -27.4

Not transfer

75.3 4.7 37.4 67.8 215.6 38.0

* Transfer: CD4+CD25+T cells transferred to BD-like mice; Difference: The difference between CD4+CD25+T cells transferred to BD-like mice and not transferred BD-like mice. The serum amount of cytokine of CD4+CD25+T cells transferred BD-like mice was subtracted from not transferred BD-like mice; Not transfer: The average amount of not transferred BD-like

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Ⅳ. DISCUSSION

Our results show the number of CD4+CD25+T cells in BD-like mice were significantly lower than both BDN and normal mice. Moreover, frequency of Treg cells in BD-like mice was lower than BDN and normal mice. It has been reported that CD4+CD25+ Treg cells are lower in autoimmune and inflammatory diseases, such as Crohn’s Disease (Ricciardelli, et al., 2008), Multiple Sclerosis (MS)(Huan, et al., 2005), and Systemic Lupus Erythematosus (Lee, et al., 2008), than in healthy control and inactive patients. Also mouse models of autoimmune and autoinflammatory, such as collagen-induced arthritis (Morgan, et

al., 2005) and experimental autoimmune encephalomyelitis (EAE)(Begum-Haque, et al.,

2008), were lower than healthy mice. But CD4+CD25+ Treg cells in rheumatoid arthritis was not different (Lee, et al., 2008) or higher than healthy controls (Han, et al., 2008). Interestingly, Nanke et al. reported that the percentage of Treg cells among CD4+T cells from BD patients with an ocular attack were significantly decreased before ocular attack compared with those after ocular attack (Nanke, et al., 2008). Treg cells of BD-like mice with symptoms maintain lower levels than normal mice.

IL-2 and TGF-β are T cell growth factors and these can induce CD4+CD25+ Treg cells (Zheng, et al., 2007). In addition, these cytokines play important roles in the development, survival, and function of CD4+CD25+ Treg cells (Zheng, et al., 2007). Our experiments have shown these cytokines effectively expand CD4+CD25+ Treg cells. Furthermore CD4+CD25+T and Treg cell levels by primary culture of normal splenocytes

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were higher, compared to BD-like and BDN mice. Marguti et al. reported that bone marrow-derived DCs from BALB/c mice were able to promote expansion of CD4+CD25+ Treg cells

in vitro. Both mature and immature DCs are capable of expanding the CD4+CD25+ Treg

cell population (Marguti, et al., 2008). Our results show the frequencies of CD4+CD25+T and Treg cells from the co-culture with CD4+T cells isolated from normal splenocytes and normal mDC was higher than CD4+T cells from splenocytes of BDN and BD-like mice. But, the frequencies of CD4+CD25+T and Treg cells from co-culture with normal CD4+T cells and each group of mDCs (normal, BDN, and BD-like) were not different among groups. According to Fig. 2 and Fig. 3, we show the proliferation of CD4+CD25+T and Treg cells from normal healthy mice was higher than from BDN and BD-like mice. The proliferation of CD4+CD25+T and Treg cells was more dependent to CD4+T cell than DCs.

Increased regulatory T cell numbers or functions have been associated with improvement of inflammatory symptoms, such as MS, EAE, and diabetes in mice and humans (Baecher-Allan, et al., 2004; McGeachy, et al., 2005; Kukreja, et al., 2002; Sakaguchi , 2004; Viglietta, et al., 2004). CD4+CD25+ Treg cells have a role in maintaining immunologic self-tolerance and lead to the decrease of spontaneous development of various autoimmune diseases (Takahashi, et al., 1998). Therefore we forecasted improvement of symptoms of BD-like mice by transfer with CD4+CD25+T cells on the basis of the foundation of our results. After transfer with CD4+CD25+T cells, the frequencies of

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CD4+CD25+T cells. According to the amount of transferred CD4+CD25+T cells, frequencies of CD4+CD25+T and Treg cells were changed in splenocytes of BD-like mice. We show CD4+CD25+T and Treg cells were up-regulated by transfer with CD4+CD25+T cells. After CD4+CD25+T cells transfer, we observed a change of symptoms in BD-like mice. Two weeks after transfer, the symptoms of BD-like mice showed improvement and also the disease severity score was decreased compared to transfer of CD4+CD25-T cells to BD-like mice. It has been reported that disease severity score decreased by transfer with CD4+CD25+T cell in EAE (Zhang, et al., 2004) and CIA (Morgan, et al., 2005). We will do further research at a later date to confirm direct effect or proliferation. The improvement of symptoms and increase of Treg in peripheral tissues occurred after CD4+CD25+T cells transfer. Therefore, adoptive transfer definitely affected the improvement of BD-like symptoms.

After transfer of CD4+CD25+T cells, cytokine expression profiles were compared with control groups. Our results show the expression of TGF-β and IL-10 was increased after transfer compared to not transferred BD-like mice. Zheng et al. reported that CD4+CD25+T cells cultured, with TGF-β and IL-2, changed cytokines levels of IL-10 and TGF-β. IL-10 and TGF-β were increased but IFN-γ was decreased (Zheng, et al., 2004). According to a paper of Chai et al . Treg cells have up-regulated TGF-β and IL-10 (chai, et

al., 2008). We show CD4+CD25+T cell transfer regulated the improvement of symptoms

through up-regulation of TGF-β and IL-10. TNF-α, and IFN-γ levels were decreased after transfer compared to not transferred BD-like mice. IFN-γ is a Th1 cytokine (Mosmann, et al., 1989). TNF-α is a mediator of inflammatory and immune functions. Th17 cells are highly

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pro-inflammatory cells and emerging data suggest that they orchestrated tissue inflammation and organ-specific autoimmune diseases (Bettelli, et al., 2007). IL-17 is a Th17 cell mediated cytokine and IL-6 inhibits CD4+CD25+T cell regulatory functions (Wan, et al., 2007). IL-6 is highly elevated in the culture supernatants of peripheral blood mononuclear cells (PBMC) of patients with active Behcet’s disease (Yamakawa, et al., 1996). From our results, IL-17 and IL-6 proteins were decreased by transfer with CD4+CD25+T cells to BD-like mice and indicated similar levels of BDN mice. Foxp3 inhibits RORγt-mediated IL-17A mRNA transcription (Ichiyama, et al., 2008). mRNA levels of ROR-γt, transcription factor

of Th17 cells, in 3 x 105CD4+CD25+T cells transferred BD-like mice were lower than not

transferred BD-like mice. The levels of IL-17A (IL-17) and IL-17F produced by Th-17 cells

in 3 x 105CD4+CD25+T cells transferred like mice were lower than not transferred

BD-like mice. Also Th17 cell-mediated cytokines and transcription factors were inhibited by CD4+CD25+ Treg cells transfer.

In summary, frequency of CD4+CD25+ Treg cells in BD-like mice was lower than BDN and normal mice. In vitro, the proliferation of splenocytes from BD-like mice was doen-regulated compared to BDN. Adoptive transfer of CD4+CD25+T cells in BD-like mice improved symptoms and up-regulated CD4+CD25+T cells from splenocytes. After transfer of CD4+CD25+T cells, IL-10 and TGF-β expressions increased but IFN-γ, TNF-α, IL-6, and IL-17 expressions decreased compared with not transfer BD-like mice.

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Ⅴ. CONCLUSION

We confirmed the frequency of CD4+CD25+T cells in BD-like mice was lower than BDN mice. CD4+CD25+T and Treg cell levels by primary culture of splenocytes were lower in BD-like mice compared to BDN mice. The frequencies of CD4+CD25+T and Treg cells in co-cultures from normal CD4+T cells isolated from splenocytes with normal mDC was higher than CD4+T cells from splenocytes of BDN and BD-like. In vivo, the frequency

of CD4+CD25+T cells were higher in transfer with 3 x 105CD4+CD25+T cells in BD-like

mice compared to transfer with 3 x 103and 3 x 104CD4+CD25+T cells. In addition, transfer

with CD4+CD25+T cells to BD-like mice improved the BD-like symptoms. The severity scores were accordingly decreased compared to transfer of CD4+CD25-T cells to BD-like

mice. After 3 x 105 CD4+CD25+T cells transfer, the severity score of BD-like mice was

decreased compared to before the transfer. The serum protein levels of TGF-β and IL-10 in

BD-like mice transferred with 3 x 105 CD4+CD25+T cells were higher, but IFN-γ, TNF-α,

IL-6, and IL-17 were lower than not transferred BD-like mice. The mRNA level of TGF-β was also affected in the transfer of CD4+CD25+T cells. TGF-β was higher than not transferred BD-like mice. Whereas IFN-γ, TNF-α, IL-17A, IL-17F, and ROR-γt of transferred with CD4+CD25+T cells were lower than not transferred BD-like mice.

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수치

Fig. 1. The frequencies of CD4+CD25+T and Treg cells in splenocytes of BDN
Fig. 7. The comparison of the severity score before and after transfer of
Table 1. The sequence of primers used for RT-PCR ··················································· 9
Table 1. The sequence of primers used for RT-PCR.
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