CD + 4 CD + 25 T The role of CD + 4 CD + 25 Regulatory T cells in the pathogenesis of vitiligo

CD + 4 CD + 25 T The role of CD + 4 CD + 25 Regulatory T cells in the pathogenesis of vitiligo 7000064 2008 5 1

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CD + 4 CD + 25 CD + 4 CD + 25 Vitiligo patients II

8+T DETACHaBEAD CD8+T Miltenyi CD4+T CD25 CD4+T CD4+CD25+T CD4+CD25-T 3 CD4+CD25+ T 4 CD4+CD25+ T 5 RNA RT-PCR Foxp3CTLA-4 GITR Western-blot Foxp3 6 SPSS11.0 ± P<0.05 1 39 CD4+CD25+ T CD4+CD25+ T CD4+CD25+ T NB-UVB 3 CD4+CD25+ T CD4+CD25+ Treg CD4+CD25+ T CD + + 4 CD 25 CD + + 4 CD 25 CD + + 4 CD 25 III

CD + + 4 CD 25 CD + + 4 CD 25 CD + + 4 CD 25 IV

The role of CD + 4 CD + 25 Regulatory T cells in the pathogenesis of vitiligo NameWu Yan-Hong SupervisorYang Hui-lan ABSTRACT Vitiligo is a common dermatological disorder characterized by milky-whi te depigmented macules devoid of identiiable melanocytes. In the recent year s there is a gradual increase in the incidence of vitiligo. The cause of vitilig o is probably associated with genetic and environmental factors. However, t he exact cause of the illness remains unknown, but several hypotheses about i ts pathogenesis are advanced. The hypothesis of autoimmunity is subjected to more and more concern recently. An exciting new area of immunologic interest has been the identification of immune-regulatory cells (Tregs) and their role in maintaining immune tole rance. The CD 4 + Tregs come from thymus gland, accounting for 5-10% of t he CD + 4 T cells in the peripheral blood. Naturally occurring CD + 4 Tregs, the majority of which express CD 25, can suppress the activation, proliferation an d differentiation of self-activated T cells. Foxp3a transcriptional factor repor ted recently, is a member of the forkhead family of transcriptional regulators. Foxp3 constitute expresses in CD + 4 CD + 25 Tregs and mainly in thymus gland, spleen and lymph nodes. CD + 4 CD + 25 Tregs have two major features, includ ing immunotolerance and immunosuppression. They are tolerant to the stimul ation of the high concentration of IL-2, solid-phase coating or soluble anti-cd i

ABSTRACT 3 antibody, and the joint stateof anti-cd3 and anti-cd28 antibodies with no s ecretion of IL-2. The immunosuppression of Tregs shows that the activation of Tregs requires the stimulation of T-cell receptors and some other signals. Once activated, their suppression is not specific. They can cause local immu nosuppression by direct interaction between cells and the secretion of IL-10 a nd TGF-band this immunosuppression has no restriction to MHC. After the activation of Tregs, the expression of CTLA-4 is increased. The anti-ctla-4 antibody can be used to block the immunosuppression of Tregs and it is gen erally believed that Tregs play effects by cell interactions. The glucocorticoid -induced tumor necrosis factor receptor(gitr), the 18 th member of TNFRSF, i s expressed on the surface of Tregs, with the ligand GITRL. GITR and CTL A-4 can increase the expression of Foxp3. The discovery of Foxp3 as a spe cific marker of Tregs has led to an explosion of research in the function of Tregs. Although the role of Tregs in the pathogenesis of vitiligo was reporte d overseas, their results are inconsistent. However, there is no report of this f ield in China yet. Objectives: To study the role of CD + 4 CD + 25 Regulatory T cells in the pathogenesis of vitiligo Methods: 1. The blood was taken from vitiligo patients and normal control and density centrifugation was used to separate the PBMC. 2. CD + 8 T cells were separated by Dynabeads, and Militenyi immunity beads were applied to separate the CD + 4 CD + 25 T cells and CD + 4 CD - 25 T cells. 3. The number of CD + 4 CD + 25 Tregs in PBMC of vitiligo patients and the normal control was analyzed by flow cytometry. 4. Flow cytometry was applied to analyze the number of CD + 4 CD + 25 Tregs in ii

PBMC of vitiligo patients both in active stage and post-treatment by NB-UVB. 5. RT-PCR was used to analyze the mrna expression level of Foxp3, CTLA-4 and GITR,and Western-blot was applied to analyze the protein expression level of Foxp3. 6. Statistics analysis: The program SPSS11.0 was used to analyze all the statistical data. When P<0.05, the difference is significant. Results: 1. The number of CD 4 + CD 25 + Tregs in peripheral blood mononuclear cells(pbmc) of 39 vitiligo patients and normal control was analyzed by flow cytometry. The results showed that the percentage of CD + 4 CD + 25 Tregs in PBMC of vitiligo patients in active stage was lower than that of normal control, and the difference is significant. After treated by NB-UVB, the percentage of CD + + 4 CD 25 Tregs of vitiligo patients was higher than that of vitiligo patients in the active stage and the difference is also significant. These results suggested that the CD + + 4 CD 25 Regulatory T cells might play an important role in the pathogenesis of vitiligo. After the treatment, the percentage of CD + 4 CD + 25 Tregs was lower than the normal control, but the difference is not significant. 2. The mrna expression levels of Foxp3, CTLA-4 and GITR were analyzed by RT-PCR, and the protein expression level of Foxp3 was analyzed by Western blotting. The results showed that the expression levels of Foxp3, CTLA-4 and GITR in the patients of the active stage were lower than those of the normal control, and the difference is significant. After treated by NB-UVB, the expression level of Foxp3 of the vitiligo patients was lower than that of normal control, while difference of the number of CD + 4 CD + 25 Regulatory T cells between the two groups is not significant. These results demonstrated that after the treatment, the number of CD + + 4 CD 25 iii

ABSTRACT Regulatory T cells had been normalized but their function has possibly been changed. Conclusions: To investigate the role of CD + 4 CD + 25 Tregs in the pathogenesis of vitiligo, this study indicated that after the NB-UVB treatment, the number of CD + 4 CD + 25 Tregs of vitiligo patients had been restored but the expression level of Foxp3 was lower than that of normal control. It suggested that the function CD + 4 CD + 25 Tregs had possibly been changed after the treatment, and the CD + 4 CD + 25 Regulatory T cells might play an important role in the pathogenesis of vitiligo. Key wordsvitiligo, CD 4 + CD 25 + Regulatory T cells, Foxp3, flow cytometry, RT-PCR, Western blot iv

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