Study On Analysis Of Peripheral Skin-homing CD8~+T Cells From Patients With Non-segmental Vitiligo And The Inhibition Effects Of Apigenin On These Cells

Vitiligo is a depigmentation disorder affecting around1%to2%of the generalpopulation. It can be separated into segmental and non-segmental types. The latter is themost common, and generally described as having an autoimmune etiology. Recentworks suggest that CD8~+T cells play an important role in the pathogenesis ofvitiligo.The migration of circulating T lymphocytes to the skin is not a random process,and the cutaneous lymphocyte-associated antigen (CLA) antigen is considered a homingreceptor for effector memory T cells with tropism for the skin. Recent studies suggestthat the CLA~+CD8~+T cells may be the autoreactive cells in vitiligo, and the studies onthe characterization of peripheral CLA~+CD8~+T cells will be helpful to reveal theimmune abnormalities of vitiligo.Regulatory T cells (Tregs) play a crucial role in the maintenance of peripheraltolerance via suppression of self-reactive T cells. More and more studies suggested thatan attenuated suppressive function of peripheral Tregs on self-reactive T cells may leadto self-reactive immune responses. We hypothesized that there may be a decreasedinhibition function of peripheral Tregs on autologous CLA~+CD8~+T cells in thepathogenesis of vitiligo.Traditional Chinese medicines (TCMs) were used for the treatment of vitiligo witha long history, but the therapeutic mechanism remains to be studied. Recently, someingredients of Chinese medicines which were used to treat vitiligo in TCM prescriptionswere reported for their inhibition to T cell. Thus, studies on ingredients of TCMs withsuppressive function to self-reactive CLA~+CD8~+T cells may provide experimental basisfor the application of these ingredients in the treatment of vitiligo. Objective:1. To investigate the frequencies of peripheral CD8~+T cells and CLA~+CD8~+T cells,and their expression of cytotoxic molecules (perforin、granzyme-B and FasL), andmigration-related molecules (CCR4and CCR10) in non-segmental vitiligo patients.2. To investigate the inhibition effects of peripheral Tregs on proliferation ofautologous CLA~+CD8~+T cells in non-segmental vitiligo patients and the expressionlevels of functional molecules on Tregs.3. To seek for ingredients of TCMs with suppressive effects on self-reactiveCLA~+CD8~+T cells, and investigate the mechanisms.Methods:1. Twenty seven patients (15with active and12with stable non-segmental vitiligo)and15healthy control volunteers were enrolled in the study. The frequencies of CD8~+T cells and CLA~+CD8~+T cells in peripheral blood of participants were investigated byflow cytometry, and their expression levels of cytotoxic molecules (perforin,granzyme-B and FasL) and chemokine receptors (CCR4, CCR10) were evaluated also.2. In7active non-segmental vitiligo patients,6stable patients and6controls, theexpression levels of functional molecules on Tregs in peripheral blood of participantswere investigated by flow cytometry. The proliferative responses of peripheralCLA~+CD8~+T cells were assessed in the absence or presence of autologous Tregs usingCFSE.3. Nine patients with active non-segmental vitiligo patients were enrolled in thispart. Five ingredients of TCMs, including apigenin, wogonin, kaempferol, peoniflorinand geniposide, were tested. To test the cytotoxicity of the ingredients, the viability ofcells was evaluated by the CCK-8assay. And the inhibition effects and relatedmechanisms of these ingredients on CLA~+CD8~+T cells were investigated.Results:1. Detection of frequency and hyper-activated cytotoxic functions of peripheralCD8+CLA~+T cells from active vitiligo patients1) There was no significant difference in the percentages of peripheral CD8~+T cellsamong the active vitiligo, stable vitiligo and control group. The expression levels ofcytotoxic molecules (perforin、granzyme-B and FasL) were of no significant differenceamong the three groups.2) The frequencies of peripheral CD8+CLA~+T cells were significantly higher in theactive vitiligo compared with the stable vitiligo and control group, and a positive correlation was found between Vitiligo Area Scoring Index (VASI) and the frequenciesof CLA~+CD8~+T cells. The expression levels of CCR4on CD8~+T cells were significantlyhigher in the active than in the stable vitiligo or control group, but the expression levelsof CCR10on CD8~+T cells were of no significant difference among the three groups.3) Both the expression levels of perforin and granzyme-B on peripheralCD8+CLA~+T cells were significantly higher in the active vitiligo compared with thestable vitiligo and control group, but the expression levels of FasL on CLA~+CD8~+T cellswere of no significant difference among the three groups. And the expression levels ofCCR4and CCR10on CLA~+CD8~+T cells were of no significant difference among thethree groups.2. Detection of suppressive function of Tregs on the proliferation of autologousCLA~+CD8~+T cells1) The expression levels of cytotoxic T lymphocyte-associated antigen-4(CTLA-4)on peripheral Tregs were significantly higher in the active vitiligo compared with thestable vitiligo and control group, but the expression levels of lymphocyte activationgene-3(LAG-3)、CD39and CD73were of no significant difference among the threegroups.2) The percentages of proliferating peripheral CLA~+CD8~+T cells stimulated withanti-CD3/CD28mAbs were of no significant difference among the three groups.However, after cocultured with autologous Tregs, the percentages of proliferatingperipheral CLA~+CD8~+T cells were significantly higher in the active vitiligo comparedwith the stable vitiligo and control group.3) Tregs showed an attenuated suppressive function on the proliferation ofautologous CLA~+CD8~+T cells in the active vitiligo compared with the stable vitiligo andcontrol group.3. Analysis of inhibition effects of ingredients of TCMs with functions ofimmuno-regulation on peripheral CD8+CLA~+T cells from active vitiligo patients1) The proliferative responses of anti-CD3/CD28mAbs stimulated peripheralCLA~+CD8~+T cells from active non-segmental vitiligo patients were inhibited obviouslyby apigenin in a dose-dependent manner, while Wogonin, kaempferol, peoniflorin andgeniposide showed no inhibition effects.2) The secretion of IL-2of peripheral CLA~+CD8~+T cells was inhibited obviously.However, the production of IFN-γ and TNF-α was not effected by apigenin.3) Apigenin induces peripheral CLA~+CD8~+T cell cycle arrest at G0/G1phase. Conclusion:1. Patients with active non-segmental vitiligo have a higher frequency of peripheralCD8+CLA~+T cells with hyper-activated cytotoxic functions (perforin and granzyme-B),which may be involved in the pathogenesis of non-segmental vitiligo.2. Tregs showed an attenuated suppressive function on the proliferation ofautologous CLA~+CD8~+T cells, which may be responsible for the increased population ofperipheral CLA~+CD8~+T cells in the active non-segmental vitiligo.3. Apigenin showed an obvious inhibition on the proliferation of activatedperipheral CD8+CLA~+T cells, which provides experimental basis for the application ofapigenin in the treatment of vitiligo.