The role of DOCK8 in autoimmunity

 描述(由申请人提供)：DOCK8缺乏症的特征是反复发生鼻窦肺部感染、慢性病毒性皮肤感染、嗜酸性粒细胞增多和IgE水平升高。DOCK8患者也易患自身免疫性；他们会患上自身免疫性溶血性贫血、结肠炎、硬化性结肠炎和血管炎。我们发现DOCK8缺乏会导致患者自身抗体的产生，患者的调节性T(Treg)细胞数量减少，体外抑制活性受损(Janssen等人)。J过敏性Clin免疫(2014年出版)。此外，我们有初步数据表明，Dock8-/-小鼠产生自身抗体，Treg数量和体外抑制能力受损。此外，表达Foxp3的Treg细胞中存在选择性Dock8缺陷的小鼠在早期就会出现自发性的脾肿大和淋巴结病。 我们的总体假设是DOCK8缺乏会导致自身反应性淋巴细胞的产生。我们将使用小鼠模型来验证这一假设，并在可能的情况下利用DOCK8缺陷患者的样本来证实我们在人类上的发现。为了验证Dock8缺陷小鼠会随着年龄的增长而患上自身免疫性疾病的假设，我们将对Dock8-/-小鼠进行老化，并进行一系列的自身抗体产生和自身免疫性疾病的体征检查。如果Dock8-/-小鼠没有发生自发性自身免疫性疾病，我们将考虑用TLR激动剂诱发疾病。同时，我们将检查Dock8-/-小鼠在自身免疫倾向背景下的加速疾病。接下来，我们将使用已经产生的Dock8条件基因敲除小鼠，研究T和B细胞固有的Dock8缺陷在发展自身免疫中的作用。最后，我们有初步数据表明，Dock8对于Treg细胞的数量和抑制活性是重要的。我们将检测Dock8缺陷小鼠Treg细胞的发育、存活和稳定性，并阐明这些缺陷是否是Treg细胞固有的。此外，使用几种体内自身免疫/炎症模型，我们将测试Dock8缺陷的Treg细胞抑制功能。总而言之，这些研究应该会对DOCK8缺乏患者的自身免疫发展机制得出答案。 我们已经在内部培育了Dock8缺陷小鼠。这些小鼠的Dock8基因座在外显子9上有一个突变，该突变模仿在一名DOCK8缺乏症患者身上发现的无意义突变，该患者不表达任何DOCK8蛋白。此外，我们还设计了Dock8Flox/FLOX条件性基因敲除小鼠。我们已经用这些小鼠培育了CD4-Cre和Foxp3-YFP-Cre小鼠，以检测Dock8在T细胞和Treg细胞内在方式中的作用。我们正在将Dock8Flox/FLOX小鼠与mb-1-Cre交配，以检测B细胞的内在效应。这些小鼠将使我们能够检查Dock8在产生自身抗体和自身免疫中的细胞内在作用。我们还可以通过参加国际免疫缺陷联盟(ICID)来接触DOCK8缺乏患者的独特队列。通常，通过ICID转介的患者在波士顿儿童医院接受评估和治疗，这使我们能够获得血液样本，以验证我们的小鼠研究结果。在这里，我们提出了有希望的数据，这些数据构成了这一科学应用的基础。通过利用小鼠模型和患者样本，我们应该能够以一种有意义的方式深入研究这些问题，以解释人类疾病。詹森博士在杜克大学获得免疫学医学博士和博士学位。她的研究生研究重点是淋巴细胞信号。在实习期间，詹森博士接受了临床免疫学和风湿学方面的培训。她目前在波士顿儿童医院接受免疫科主任Raif Geha博士的指导。在他的指导下，Janssen博士发表了两篇关于DOCK8缺乏患者的淋巴细胞亚群和TLR4在IgE类转换中的作用的第一作者手稿。此外，她的两篇第一作者手稿最近被《过敏和临床免疫学杂志》接受，内容是DOCK8和LRBA缺陷患者的自身免疫和受损的调节性T细胞的发展。 K08申请的成功资助将使Janssen博士有能力继续这些研究，并进一步发展她作为内科科学家的职业生涯。她会继续 有机会参与波士顿儿童基金会、哈佛医学院和哈佛促进会提供的各种职业发展机会。詹森博士的目标是在她的导师、顾问委员会和哈佛医学院经验丰富的教职员工的指导下，在未来五年内实现研究的独立性。她希望继续从自己的临床经验中探索自身免疫和免疫失调方面的新问题。

 DESCRIPTION (provided by applicant): DOCK8 deficiency is characterized by recurrent sinopulmonary infections, chronic viral skin infections, eosinophilia, and elevated IgE levels. DOCK8 patients are also predisposed to autoimmunity; they develop autoimmune hemolytic anemia, colitis, sclerosing colitis, and vasculitis. We found that DOCK8 deficiency in patients leads to the development of autoantibodies, and patients have decreased numbers of regulatory T (Treg) cells and impaired Treg cell in vitro suppressive activity (Janssen et al. J Allergy Clin Immunol (2014) in press). In addition, we have preliminary data that Dock8-/- mice develop autoantibodies and have impaired Treg numbers and in vitro suppressive ability. Furthermore, mice with a selective Dock8 deficiency in Foxp3 expressing Treg cells develop spontaneous splenomegaly and lymphadenopathy at an early age. Our overall hypothesis is that DOCK8 deficiency leads to the generation of autoreactive lymphocytes. We will test this hypothesis using mouse models, and when possible utilize samples from DOCK8 deficient patients to confirm our findings in humans. To test the hypothesis that Dock8 deficient mice develop autoimmune disease with aging, we will age Dock8-/- mice and do serial examinations for autoantibody production and signs of autoimmune disease. If Dock8-/- mice do not develop spontaneous autoimmune disease, we will look at inducing disease with TLR agonists. Simultaneously, we will examine Dock8-/- mice for accelerated disease in the context of autoimmune prone backgrounds. Next, we will examine the role of T and B cell intrinsic Dock8 deficiency in developing autoimmunity, using Dock8 conditional knock-out mice that have already been generated. Finally, we have preliminary data that Dock8 is important for Treg cell number and suppressive activity. We will examine the development, survival, and stability of Treg cells in Dock8 deficient mice and elucidate whether these defects are Treg cell intrinsic. In addition, using several in vivo models of autoimmunity/inflammation, we will test Dock8 deficient Treg cell suppressive function. Together, these studies should yield answers regarding the mechanism of developing of autoimmunity in DOCK8 deficient patients. We have generated Dock8 deficient mice in-house. The Dock8 locus in these mice have a mutation in exon 9 that mimics a nonsense mutation found in a DOCK8 deficient patient who does not express any DOCK8 protein. In addition, we have engineered Dock8flox/flox conditional knock-out mice. We have already bred these mice with Cd4-Cre and Foxp3-YFP-Cre mice to examine role of Dock8 in a T cell and Treg cell intrinsic manner. We are in the process of mating the Dock8flox/flox mice with mb-1-Cre to examine B cell intrinsic effects. These mice will allow us to examine cell intrinsic roles of Dock8 in the generation of autoantibodies and autoimmunity. We also have access to a unique cohort of DOCK8 deficient patients through participation in the International Consortium of Immunodeficiency (ICID). Often patients referred through the ICID are evaluated and treated at Boston Children's hospital, and this allows us to obtain blood samples for validation of our mouse findings. Here, we present promising data that forms the basis for this scientific application. Through the utilization of thee mouse models and patient samples, we should be able to delve into these questions in a meaningful way to explain the human disease. Dr. Janssen received her M.D. and Ph.D. in Immunology at Duke University. Her graduate studies focused on lymphocyte signaling. During her residency and fellowship, Dr. Janssen acquired training in clinical immunology and rheumatology. She is currently mentored by Dr. Raif Geha, Chief of Immunology, at Boston Children's Hospital. Under his guidance, Dr. Janssen published two first author manuscripts on lymphocyte subsets in DOCK8 deficient patients and the role of TLR4 in IgE class switching. In addition, she had two first author manuscripts recently accepted to the Journal of Allergy and Clinical Immunology on the development of autoimmunity and impaired regulatory T cells in DOCK8 and LRBA deficient patients. Successful funding of this K08 application will give Dr. Janssen the ability to continue these studies, as well as to further develop her career as a physician scientist. She will continue to have the opportunity to participate in the various career development opportunities provided by Boston Children's, the Harvard Medical School, and the Harvard Catalyst. Dr. Janssen's goal over the next five years is to progress to research independence under the guidance of her mentor, advisory committee, and the experienced faculty at Harvard Medical School. She would like to continue to draw from her clinical experience to explore new questions in autoimmunity and immune dysregulation.