Therapeutic treatment of EAT by inducing dendritic cell

描述(申请人提供)：实验性自身免疫性甲状腺炎(EAT)的特征是浸润性淋巴细胞最终破坏甲状腺，导致甲状腺功能减退。产生干扰素-γ、肿瘤坏死因子-α和白介素12的CD4+T细胞在发病机制中起关键作用。在最近的一项研究中，我们发现，与甲状腺球蛋白免疫的对照组相比，Flt3-L免疫的小鼠出现了更严重的EAT，其特征是大量甲状腺淋巴细胞浸润，IL-2和干扰素-γ的产生。相比之下，GM-CSF治疗的小鼠未能形成EAT，并显示出CD4+CD25+调节性T细胞(Tregs)显著增加。用MTG体外激活这些小鼠的淋巴细胞，产生更高水平的IL-4和IL-10。中和IL-10，但不中和IL-4，并去除这些培养物中的Tregs，可以恢复MTG特异性T细胞的增殖，以及IL-2和干扰素-γ的产生。此外，过继转移Tregs给MTG免疫的小鼠抑制了EAT，而接种抗IL-10受体抗体则逆转了GM-CSF诱导的抑制，导致了EAT。这些结果表明，抑制EAT是由Tregs介导的，很可能是通过促进IL-10的产生来实现的。基于这些结果，我们推测：“用GM-CSF选择性激活CD8a-树突状细胞可以激活CD4+CD25+调节性T细胞，并使正在进行的抗甲状腺球蛋白免疫反应偏向Th2型，从而抑制实验性自身免疫性甲状腺炎的发生和/或进展。”在AIM-1中，将测试GM-CSF在再次暴露于MTG时提供长期抗原特异性保护的有效性。在AIM-2中，我们将检测CD8a-和CD8a+DC经历成熟、捕获和呈递抗原以及产生细胞因子的能力，并确定MTG特异性T细胞表型和细胞因子谱。将测试DC和T细胞在WT和IL-10-/-小鼠体内和体外抑制MTG特异性反应的能力。在AIM-3中，我们将检测GM-CSF、DC和Tregs对WT和SCID小鼠甲状腺细胞MHC和B7分子表达、ARC功能和对Fas诱导的凋亡敏感性的影响。这些研究将为GM-CSF抑制EAT的作用模式提供重要的见解。

DESCRIPTION (provided by applicant): Experimental Autoimmune Thyroiditis (EAT) is characterized by eventual destruction of thyroid by infiltrating lymphocytes resulting in hypothyroidism. CD4+ T cells that produce IFN-gamma, TNF-alpha and IL-12 are critical for the pathogenesis. In a recent study, we showed that relative to thyroglobulin (mTg) immunized controls, mice treated with Flt3-L developed a more severe EAT characterized by massive thyroid lymphocytic infiltration, and IL-2 and IFN-gamma production. In contrast, GM-CSF treated mice failed to develop EAT, and showed a significant increase in CD4+CD25+ regulatory T cells (Tregs). Activation of lymphocytes from these mice with mTg in vitro yielded higher levels of IL-4 and IL-10. Neutralization of IL-10, but not IL-4, and depletion of Tregs from these cultures restored mTg specific T cell proliferation, and IL-2 and IFN-gamma production. Further, adoptive transfer of Tregs to mTg immunized mice suppressed EAT while, inoculation of anti-IL-10 receptor Ab reversed GM-CSF induced suppression resulting in EAT. These results showed that suppression of EAT was mediated by Tregs most likely through enhanced production of IL-10. Based on these results we hypothesize that "selective activation of CD8a- dendritic cells using GM-CSF can activate CD4+CD25+ regulatory T cells and skew ongoing anti-thyroglobulin immune responses in favor of Th2 type, with consequent suppressive effects on the development and/or, progression of experimental autoimmune thyroiditis." In Aim-1, efficacy of GM-CSF to confer long-term antigen specific protection upon re-exposure to mTg will be tested. In Aim-2, we will test the ability of CD8a- and CD8a+ DCs to undergo maturation, capture and present the antigen, and produce cytokines, and determine the mTg specific T cell phenotype and cytokine profiles. Both DCs and T cells will be tested for their ability suppress in vitro and in vivo mTg specific responses in WT and IL-10-/- mice. In Aim-3, we will test the effects of GM-CSF, DCs and Tregs on the expression of MHC and B7 molecules, ARC function, and sensitivity to Fas induced apoptosis of thyrocytes from WT and SCID mice. These studies will provide significant insights into the mode of action of GM-CSF in suppressing EAT.