Therapeutic treatment of EAT by inducing dendritic cell

描述（由申请方提供）：实验性自身免疫性甲状腺炎（EAT）的特征是浸润淋巴细胞最终破坏甲状腺，导致甲状腺功能减退。产生IFN-γ、TNF-α和IL-12的CD 4 + T细胞对于发病机制至关重要。在最近的一项研究中，我们表明，相对于甲状腺球蛋白（mTg）免疫对照组，用Flt 3-L处理的小鼠发生了更严重的EAT，其特征在于大量的甲状腺淋巴细胞浸润，以及IL-2和IFN-γ的产生。相比之下，GM-CSF处理的小鼠未能发展EAT，并且显示出CD 4 + CD 25+调节性T细胞（T细胞）的显著增加。在体外用mTg活化来自这些小鼠的淋巴细胞产生更高水平的IL-4和IL-10。IL-10的中和，而不是IL-4的中和，以及从这些培养物中耗尽Tglutamine，恢复了mTg特异性T细胞增殖，以及IL-2和IFN-γ的产生。此外，过继性转移TlC至mTg免疫的小鼠抑制EAT，而接种抗IL-10受体Ab逆转GM-CSF诱导的抑制，导致EAT。这些结果表明，抑制EAT最有可能是通过增加IL-10的产生介导的。基于这些结果，我们假设“使用GM-CSF选择性激活CD 8a-树突状细胞可以激活CD 4 + CD 25+调节性T细胞，并使正在进行的抗甲状腺球蛋白免疫应答偏向Th 2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时赋予长期抗原特异性保护的功效。在Aim-2中，我们将测试CD 8a-和CD 8a + 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细胞两者在WT和IL-10-/-小鼠中抑制体外和体内mTg特异性应答的能力。目的-3：检测GM-CSF、DC和TCF 3对WT和SCID小鼠甲状腺细胞MHC和B7分子表达、ARC功能以及对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.