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

描述（由申请人提供）：候选人和培训环境：我正在寻求K01指导职业发展奖，以获得额外的培训，这将有助于我过渡到一个独立的科学家。我的长期研究目标是了解如何操纵T细胞受体信号可以用来改变或阻止不同的T细胞功能，这对开发T细胞介导疾病的治疗策略很有兴趣。作为Deborah Fowell博士的博士生，我确定了CD4+ T细胞分化对酪氨酸激酶Itk的要求。在这些研究过程中，我获得了细胞T细胞免疫学方面的培训，主要涉及小鼠原代CD4+ T细胞的分离、激活、分化和效应细胞因子的产生。到目前为止，我在Arthur Weiss博士实验室的博士后培训主要集中在开发和表征一种独特的酪氨酸激酶ZAP-70的实验性抑制剂。我们发现这种抑制剂系统是高度特异性的，可滴定的，并且可以快速和可逆地抑制ZAP-70激酶活性，使该系统成为调节T细胞受体信号传导强度的非常有用的工具。在我博士后培训的这一点上，我学会使用的技术和技巧非常适合于理解如何操纵T细胞受体信号来改变T细胞功能，这对于开发T细胞介导的自身免疫性疾病（如类风湿关节炎）的新治疗策略可能很重要。在自身免疫疾病模型分析，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，并过渡到独立的科学家。拟议的研究将在加州大学旧金山分校（UCSF）的Arthur Weiss博士的指导下进行。Weiss博士是一名风湿病学家，是T细胞信号转导领域的知名专家，也是一名杰出的导师，曾获得UCSF终身成就奖。此外，我将能够从我在UCSF的合作者/顾问，Abul Abbas博士和Mark Anderson博士那里获得建议，他们在自身免疫和耐受性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，是获得额外培训和自身免疫研究投入的丰富环境。研究：T细胞反应与多种自身免疫性疾病如类风湿关节炎（RA）有关。因此，阻断T细胞功能的治疗策略可能有助于治疗RA和其他T细胞介导的自身免疫性疾病。虽然抑制所有T细胞功能会阻断自身反应性T细胞反应，但更理想的策略是选择性地阻断引起病理的T细胞亚群。先前的工作和初步数据表明，酪氨酸激酶ZAP-70的抑制符合这些标准。首先，常规CD4+和CD8+的激活，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，Th17分化的CD4+ T细胞对ZAP-70的抑制明显更敏感。综上所述，这些数据表明，抑制ZAP-70可能对治疗由自身反应性Th17反应介导的自身免疫性疾病（如RA）非常有用。在本提案的Aim 1中，我的目标是确定Th17分化细胞对ZAP-70抑制的差异敏感性。首先，我将验证Th17细胞优先来源于表达相对低水平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，找出对ZAP-70抑制最敏感、对Th17分化非常重要的TCR信号通路。Aim 2将确定ZAP-70的不完全抑制或短暂抑制是否会导致随后的T细胞反应性低下，并确定驱动这一过程的潜在机制。最后，Aim 3的目的是确定ZAP-70抑制作为预防或改善胶原诱导的关节炎和实验性自身免疫性脑脊髓炎（两种th17介导的自身免疫性疾病小鼠模型）相关病理的治疗效果。我预计这些研究将首次表明，ZAP-70抑制有效地抑制自身免疫设置中的T细胞相关病理。最终，这些研究可能为T细胞受体信号如何改变细胞的分化和效应功能提供新的见解，这将对开发T细胞介导的自身免疫性疾病的新疗法具有重要意义。

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.