Inhibition of ZAP-70 to selectively attenuate autoimmune disease-inducing T cells

应聘者描述(申请人提供)：应聘者和培训环境：我正在寻求K01导师职业发展奖，以获得额外的培训，这将有助于我转变为一名独立的科学家。我的长期研究目标是了解如何利用操纵T细胞受体信号来改变或预防不同的T细胞功能，我的兴趣是开发T细胞介导的疾病的治疗策略。作为Deborah Fowell博士指导下的一名博士生，我确定了CD4T细胞分化过程中对酪氨酸激酶ITK的要求。在这些研究过程中，我接受了细胞T细胞免疫学方面的培训，主要与小鼠原代CD4T细胞的分离、激活、分化和效应细胞因子的产生有关。到目前为止，我在Arthur Weiss博士实验室接受的博士后培训主要集中在开发和鉴定一种独特的酪氨酸激酶ZAP-70试验性抑制剂。我们发现，该抑制系统具有高度的特异性，可滴定，并能快速和可逆地抑制ZAP-70激酶的活性，使该系统成为调节T细胞受体信号强度的非常有用的工具。在我博士后培训的这一点上，我已经学会使用的技术和技巧非常适合理解如何应用T细胞受体信号的操纵来改变T细胞功能，这可能对开发T细胞介导的自身免疫性疾病的新治疗策略非常重要，例如类风湿性关节炎。在分析自身免疫性疾病模型以及驱动自身反应性T细胞激活和分化的信号方面的进一步培训将使我能够制定独立的研究计划，并转变为一名独立的科学家。拟议的研究将在加州大学旧金山分校(UCSF)Arthur Weiss博士的指导下进行。韦斯博士是一位风湿病学家，T细胞信号转导领域的著名专家，也是加州大学旧金山分校终身成就导师奖的获得者。此外，我将能够从我在加州大学旧金山分校的合作者/顾问阿布·阿巴斯博士和马克·安德森博士那里获得建议，他们在自身免疫和耐受领域拥有丰富的经验和专业知识。加州大学旧金山分校的微生物学和免疫学系也受到高度重视，是为自身免疫研究获得额外培训和投入的丰富环境。研究：T细胞反应与类风湿性关节炎(RA)等多种自身免疫性疾病有关。因此，阻断T细胞功能的治疗策略可能对RA和其他T细胞介导的自身免疫性疾病的治疗有用。虽然抑制所有T细胞功能会阻止自身反应性T细胞反应，但更可取的策略是选择性地阻止导致病理的T细胞亚群。先前的工作和初步数据表明，抑制酪氨酸激酶ZAP-70可能符合这些标准。首先，激活常规的CD4和CD8，而不是调节性T细胞，需要ZAP-70的催化活性。其次，初步数据表明，与Th1和Th2分化细胞相比，经历Th17分化的CD4T细胞对ZAP-70的抑制明显更敏感。综上所述，这些数据表明，ZAP-70抑制可能对治疗由自身反应性Th17反应介导的自身免疫性疾病非常有用，如RA。在这个提案的目标1中，我的目标是确定Th17分化细胞如何对ZAP-70抑制产生不同的敏感性。首先，我将验证Th17细胞优先来自表达相对较低水平ZAP-70的细胞的假设。其次，我计划利用Th17细胞对ZAP-70抑制的敏感性增加的优势，找出对ZAP-70抑制最敏感的TCR信号通路，从而对Th17分化非常重要。目的2将确定ZAP-70的不完全或短暂抑制是否会导致随后的T细胞低反应性，并确定驱动这一过程的潜在机制。最后，目标3的目的是确定抑制ZAP-70作为一种治疗方法的有效性，以预防或改善与胶原诱导的关节炎和实验性自身免疫性脑脊髓炎相关的病理，这两种疾病是Th17介导的自身免疫性疾病的小鼠模型。我预计，这些研究将首次表明，在自身免疫的环境中，抑制ZAP-70有效地抑制T细胞相关的病理。最终，这些研究可能为T细胞受体信号如何改变T细胞的分化和效应功能提供新的见解，这对于开发T细胞介导的自身免疫性疾病的新疗法将是重要的。

DESCRIPTION (provided by applicant): Candidate and Training Environment: I am seeking a K01 Mentored Career Development Award to obtain additional training, which will facilitate my transition into an independent scientist. My long-term research goal is to understand how manipulation of T cell receptor signals can be used to alter or prevent diverse T cell functions, i the interest of developing therapeutic strategies for T cell mediated diseases. As a PhD student under the mentorship of Dr. Deborah Fowell, I determined the requirements for the tyrosine kinase Itk in CD4+ T cell differentiation. During the course of those studies, I gained training in cellular T cell immunology, mostly related to the isolation, activation, differentiation, and effecor cytokine production of primary mouse CD4+ T cells. My postdoctoral training in the laboratory of Dr. Arthur Weiss has so far focused on developing and characterizing a unique experimental inhibitor for the tyrosine kinase ZAP-70. We showed that this inhibitor system is highly specific, titratable, and can rapidly and reversibly inhibit ZAP-70 kinase activity, making this system a very useful tool for modulating the magnitude of T cell receptor signaling. At this point of my postdoctoral training, the technology and techniques that I have learned to use are well suited to understand how manipulations of T cell receptor signaling can be applied to alter T cell function, which could be important for developing novel therapeutic strategies for T cell mediated autoimmune diseases, such as rheumatoid arthritis. Further training in the analysis autoimmune disease models, and the signals that drive the activation and differentiation of autoreactive T cells will allow me to develop an independent research plan and transition into an independent scientist. The proposed studies will take place under the mentorship of Dr. Arthur Weiss at the University of California, San Francisco (UCSF). Dr. Weiss is a rheumatologist, a renowned expert in the field of T cell signal transduction, and a distinguished mentor as the recipient of te UCSF Lifetime Achievement in Mentoring award. Additionally, I will be able to get advice from my collaborators/consultants at UCSF, Dr. Abul Abbas and Dr. Mark Anderson, who have considerable experience and expertise in the fields of autoimmunity and tolerance. The Department of Microbiology and Immunology at UCSF is also highly regarded and is a rich environment for gaining additional training and input for studies of autoimmunity. Research: T cell responses are associated with multiple autoimmune diseases such as rheumatoid arthritis (RA). Therefore, a therapeutic strategy for blocking T cell function could be useful for treatment of RA and other T cell-mediated autoimmune diseases. While inhibiting all T cell function would block the autoreactive T cell response, a more desirable strategy would selectively block the subset of T cells responsible for causing pathology. Previous work and preliminary data suggest that inhibition of the tyrosine kinase ZAP-70 could fit these criteria. First, the activation of conventional CD4+ and CD8+, but not regulatory T cells, requires ZAP-70 catalytic activity. Secondly, preliminary data indicates that CD4+ T cells undergoing Th17 differentiation are substantially more sensitive to ZAP-70 inhibition as compared to Th1 and Th2 differentiating cells. Together these data suggest that ZAP-70 inhibition could be very useful for treating autoimmune diseases mediated by autoreactive Th17 responses, such as RA. In Aim 1 of this proposal, I aim to determine how Th17 differentiating cells are differentially sensitive to ZAP-70 inhibition. First, I will test the hypothesis that Th17 cells are preferentially derived from cellsthat express relatively low levels of ZAP-70. Secondly, I plan to take advantage of the increased sensitivity of Th17 cells to ZAP-70 inhibition to identify TCR signaling pathways that are most sensitive to ZAP-70 inhibition and thus highly important for Th17 differentiation. Aim 2 will determine whether incomplete or transient inhibition of ZAP-70 results in subsequent T cell hyporesponsiveness and identify potential mechanisms that drive this process. Lastly, the goal of Aim 3 is to determine the efficacy of ZAP-70 inhibition as a therapy to either prevent or ameliorate pathology associated with collagen-induced arthritis, and experimental autoimmune encephalomyelitis, two mouse models of Th17-mediated autoimmune disease. I anticipate that these studies will show for the first time that ZAP-70 inhibition effectively inhibits T cell-associated pathology in settings of autoimmunity. Ultimately, these studies may provide new insights into how T cell receptor signaling can alter the differentiation and effector functions ofT cells, which will be important for developing novel therapies for T cell-mediated autoimmune disease.