Roles of CD2 and 2B4 in invariant natural killer T (iNKT) cell functions.

描述(申请人提供)：不变自然杀伤T细胞(INKT)是先天的T淋巴细胞，参与宿主对癌症的免疫。尽管人们认识到iNKT细胞促进抗肿瘤免疫反应，但关于这些细胞如何识别和响应肿瘤细胞仍然存在许多问题。这种信息的缺乏阻碍了基于iNKT细胞的癌症免疫疗法的发展，这种疗法可能更有效。我的研究表明，原代培养的小鼠和人iNKT细胞在体外和体内都显示出强大的T细胞受体(TCR)诱导的杀瘤活性。为了阐明调节TCR诱导的iNKT细胞反应的重要机制，我最近观察到免疫球蛋白超家族受体CD2和2B4对小鼠iNKT细胞的细胞毒作用具有差异性调节作用。具体地说，CD2-/-iNKT完全不能杀死肿瘤靶点，而2b4-/-iNKT 表现出显著的细胞溶解增强。基于这些发现，我推测CD2是TCR诱导的iNKT细胞功能的正性调节因子，2B4是负性调节因子。在这个K22提案中，我将通过进一步探讨CD2和2B4在体内iNKT细胞介导的肿瘤清除(Aim 1a)和iNKT细胞免疫刺激功能(细胞因子的产生和其他免疫细胞的激活)(Aim 1b)中的作用，来建立这些新的和令人兴奋的观察结果。此外，我还将研究这些受体在人iNKT细胞激活、增殖、细胞因子产生和体外抗肿瘤反应中的作用(目标1c)。为了剖析这些分子调节iNKT细胞毒性的机制基础，我将评估CD2或2B4信号的中断如何影响iNKT细胞免疫突触的形成和成熟(目标2a)。CD2和2B4都与其配体CD48结合，但亲和力不同。此外，CD2和2B4具有不同的细胞质尾巴，可能赋予不同的信号功能。因此，我将描述CD2和2B4是否通过诱导不同的细胞内信号(目标2b)和/或通过相互竞争CD48结合(目标2c)来发挥相反的作用。由于CD1d(不变TCR的配体)和CD48(CD2和2B4的配体)在血液系统肿瘤中广泛表达，进一步阐明CD2和2B4调节TCR诱导的iNKT细胞抗肿瘤活性的机制具有重要的科学和临床意义。这些研究将有助于更好地了解iNKT细胞生物学，并为如何以治疗相关的方式利用iNKT细胞的抗肿瘤活性提供见解。 为了能够成功地完成这些研究，并促进我的科学和职业发展，我已经建立了关键的合作和一个科学咨询委员会，成员包括斯蒂芬·格鲁普博士、乔丹·奥兰治博士和Taku Kambayashi博士，他们都是各自研究领域的专家。我会利用我的智力优势和学术实力 我的导师和科学咨询委员会成员的记录，以及费城儿童医院和宾夕法尼亚大学提供的专业知识、设施和资源的强大可用性，以完成本提案中概述的培训计划。

DESCRIPTION (provided by applicant): Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that participate in host immunity to cancer. Despite the recognition that iNKT cells promote anti-tumor immune responses, many questions remain regarding how these cells recognize and respond to tumor cells. This lack of information impedes the development of novel and potentially more effective iNKT cell-based immunotherapies for cancer. My studies reveal that primary murine and human iNKT cells exhibit robust T cell receptor (TCR)-induced cytolytic activity against tumor cells in vitro and in vivo. In an attempt to elucidate the signalig mechanisms that regulate TCR-induced iNKT cell responses, I recently observed that immunoglobulin superfamily receptors, CD2 and 2B4 differentially regulate murine iNKT cell cytotoxicity. Specifically, Cd2-/- iNKTs completely failed to kill tumor targets while 2b4-/- iNKTs exhibited significantly enhanced cytolysis. Based upon these findings, I hypothesize that CD2 is a positive and 2B4 a negative regulator of TCR-induced iNKT cell functions. In this K22 proposal, I will build upon these new and exciting observations by further exploring the roles of CD2 and 2B4 in iNKT cell-mediated tumor clearance in vivo (Aim 1a) and iNKT cell immuno-stimulatory functions (cytokine production and activation of other immune cells) in vitro and in vivo (Aim 1b). Additionally, I will also examine the roles of these receptors in human iNKT cell activation, proliferation, cytokine production and anti- tumor responses in vitro (Aim 1c). To dissect the mechanistic basis by which these molecules regulate iNKT cell cytotoxicity, I will assess how disruption of CD2 or 2B4 signaling affects iNKT cell immunological synapse formation and maturation (Aim 2a). CD2 and 2B4 both bind to their ligand, CD48 but with different affinities. Furthermore, CD2 and 2B4 have different cytoplasmic tails, which are likely t confer distinct signaling functions. Therefore, I will delineate whether CD2 and 2B4 exert opposing roles by inducing distinct intracellular signals (Aim 2b) and/or by competing with one another for CD48 binding (Aim 2c). As CD1d (the ligand for the invariant TCR) and CD48 (the ligand for CD2 and 2B4) are both widely expressed on hematopoietic tumors, further elucidation of the mechanisms by which CD2 and 2B4 regulate TCR-induced iNKT cell anti- tumor activity is of significant scientific and clinical importance. These studies will facilitate a better understanding of iNKT cell biology and provide insights into how the anti-tumor activities of iNKT cells can be harnessed in a therapeutically relevant manner. To enable the successful completion of these studies and facilitate my scientific and career development, I have established critical collaborations and a scientific advisory committee that includes Drs. Stephan Grupp, Jordan Orange and Taku Kambayashi, all experts in their respective fields of investigation. I will take advantage of the intellectual strength and academic track record of my mentor and scientific advisory committee members, and the robust availability of expertise, facilities, and resources offered at The Children's Hospital of Philadelphia and the University of Pennsylvania to accomplish the training program outlined in this proposal.