Mechanism of restored immune tolerance in anti-TLR4 antibody reversal of NOD T1D

 描述(由申请人提供)：我们有令人兴奋的新数据显示，通过激动型TLR4/MD-2特异性单抗(UT18，以下称为“TLR4-Ab”)治疗，新发的1型糖尿病(T1D)(高血糖、多尿和体重减轻)在非肥胖型糖尿病(NOD)小鼠中得到逆转。接受TLR4-Ab治疗的小鼠有90%表现出临床反应(进展延迟到终末期T1D)，70%的T1D永久逆转。治疗成功的小鼠表现出胰岛炎症减轻，胰岛β细胞胰岛素染色保存。我们的方法是高度创新的，因为它针对先天免疫系统中的特定分子复合体TLR4/MD-2，以恢复适应性免疫系统的耐受性。虽然TLR4-Ab不能直接刺激T细胞，但我们发现，通过这种治疗，免疫耐受可以恢复到适应性免疫系统：来自新发糖尿病的小鼠的T细胞，然后成功地用TLR4-Ab治疗两次，不会转移疾病。这一方法对T1D和其他自身免疫性疾病具有广泛的意义：与小鼠相比，TLR4/MD-2途径在人类中进化上高度保守，为在人类中测试这一途径/免疫途径提供了理论基础。此外，已经有FDA批准的针对人类TLR4/MD-2的药物，更多的药物正在积极开发，作为癌症免疫治疗的新方法。我们认为，该途径是检测自身免疫的一种很有前途的新的治疗方法。我们建议从两个方面研究这种新方法逆转T1D和恢复获得性免疫耐受的免疫学机制。在第一个目标中，“确定TLR4-Ab治疗糖尿病小鼠恢复适应性免疫T细胞耐受的机制”，我们将解决一个令人惊讶的发现，即从新发糖尿病并成功使用TLR4-Ab治疗的小鼠中分离出的T细胞不会转移糖尿病。这些细胞是通过什么机制变得无法致病的？CD4+和CD8+T效应器(TEFs)是耐受性的或无能的，还是T调节细胞(Tregs)在数量或功能上增加了--或者Tregs和Teffs都受到了治疗的影响？我们将对抗体处理动物的细胞亚型进行研究，以确定Tregs和/或T效应细胞是否受到TLR4-Ab处理的调节。在第二个目标，TLR4-Ab治疗对T细胞受体转基因转移系统中T效应细胞和T调节细胞的作用机制的定量和功能分析，我们将使用一个严格定义的T细胞转基因转移系统来量化TLR4-Ab在自身免疫性糖尿病发病前、中和之后对特定细胞亚群的影响，并绘制TLR4-Ab治疗后转基因自身反应性T细胞的命运和功能图。了解TLR4途径介导的疾病逆转的免疫学机制具有巨大的兴趣和潜力。在两年多的时间里，这些目标应该明确地确立抗TLR4治疗的有效性和机制，以便将来应用于人类T1D和其他自身免疫性疾病。

 DESCRIPTION (provided by applicant): We have exciting new data showing reversal of new onset type 1 diabetes (T1D) (hyperglycemia, polyuria and weight loss) in nonobese diabetic (NOD) mice by treatment with an agonistic TLR4/MD-2 specific monoclonal antibody (UT18, hereafter referred to as "TLR4-Ab"). 90% of mice treated with TLR4-Ab showed a clinical response (delay in progression to endstage T1D) and 70% have permanent reversal of T1D. Successfully treated mice demonstrate decreased islet inflammation and preserved insulin staining of islet beta cells. Our approach is highly innovative because it targets a specific molecular complex, TLR4/MD-2, in the innate immune system to restore tolerance in the adaptive immune system. Although TLR4-Ab does not stimulate T cells directly, we show that immune tolerance can be restored to the adaptive immune system by this treatment: T cells from mice that had developed new onset diabetes, and then were successfully treated twice with TLR4-Ab, do not transfer disease. This approach has broad implications for T1D and other autoimmune diseases: the TLR4/MD-2 pathway is strongly evolutionarily conserved in humans compared to mice, providing a rationale to test this approach/immune pathway in humans. Moreover, there are already FDA approved drugs that target TLR4/MD-2 in humans, and more are actively being developed as a novel approach in cancer immunotherapy. We submit that this pathway is a promising new therapeutic approach for testing in autoimmunity. We propose to study the immunological mechanisms of reversal of T1D and restoration of adaptive immune tolerance achieved by this novel approach in two aims. In aim one, "Defining the mechanisms of restored adaptive immune T cell tolerance in diabetic mice treated with TLR4-Ab", we will address the surprising finding that T cells isolated from mice that had developed new onset diabetes and were successfully treated with TLR4-Ab do not transfer diabetes. By what mechanism were these cells rendered unable to cause disease? Were CD4+ and CD8+ T effectors (Teffs) rendered tolerogenic or anergic, or were T regulatory cells (Tregs) increased in quantity or function - or were both Tregs and Teffs affected by treatment? We will perform studies with cell subtypes from antibody treated animals to identify whether Tregs and/or T effector cells are modulated by TLR4-Ab treatment. In Aim Two, "Quantitative and functional analysis of the mechanistic effects of TLR4-Ab treatment on T effector and T regulatory cells in a T cell receptor transgenic transfer system", we will employ a rigorously defined T cell transgenic transfer system to quantify TLR4-Ab effects on specific cell subsets before, during and after onset of autoimmune diabetes, and map the fate and function of transgenic autoreactive T cells after TLR4-Ab treatment. Understanding the immunological mechanisms of disease reversal mediated by the TLR4 pathway is of tremendous interest and potential. These aims performed over two years should definitely establish the efficacy and mechanisms of anti-TLR4 treatment for future application to human T1D and other autoimmune diseases.