CD4+T cells from normal, BDN, and BD-like splenocytes were co-cultured

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.

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%).

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 2.7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.

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 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).

After transfer with 3 x 10⁵ CD4+CD25+T cells in BD-like mice, the IL-10 and TGF-β

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 10⁵CD4+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 10⁵CD4+CD25+T cell transferred BD-like mice than not transferred BD-like mice by RT-PCR and ELISA. By ELISA, the TNF- level

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-

in spleen tissues was detected only in BD-like mice (Fig 8G).

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 BD-like mice (n=16) compared to 33.4±7.2 pg/ml for the transferred with 3 x 10⁴

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 10⁵CD4+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.

A B

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, revealed by RT-PCR. Lane 1: BD-like mice, Lane 2: BDN, Lane 3: transferred with 3 x 10⁴

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 detected higher than not transferred BD-like mice.

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

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.

A B

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

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

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

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

문서에서 단순포진바이러스로 유도된 베체트병 마우스모델에서 CD4+CD25+ 조절 T 세포의 역할 (페이지 28-0)