Mechanisms of invariant NKT cell-mediated in vivo anti-tumor responses

描述(由申请者提供)：这份建议书描述了一个为期4年的培训计划，以发展我在儿科传染病方面的学术生涯。我已经完成了费城儿童医院(CHOP)普通儿科的正式住院医师培训和儿科传染病专科研究员培训，现在正在扩展我在NKT细胞生物学和肿瘤免疫学方面的科学技能，并在免疫能力强和免疫受损的宿主的传染病方面发展我的临床技能。金·尼科尔斯博士和加里·科雷茨基博士将指导我的科学发展。尼科尔斯博士是CHOP遗传性癌症处置计划的主任，也是宾夕法尼亚大学(宾夕法尼亚大学)儿科副教授，是NKT细胞生物学方面的专家。尼科尔斯博士在发展研究员、博士后实习生和初级教员的职业生涯方面有着越来越多的记录。为了让我的指导团队尽可能强大，尼科尔斯博士与宾夕法尼亚大学医学部研究副主席兼首席科学官科雷茨基博士和艾布拉姆森家族癌症研究所信号转导项目主任科雷茨基博士合作。科雷茨基博士是淋巴细胞生物学和T细胞信号转导方面的国际权威，在成功指导众多学生、博士后研究员和初级教职员工方面拥有丰富的经验。为了进一步促进我的科学发展，我组成了一个由乔丹·奥兰治博士、斯蒂芬·格鲁普博士和杰弗里·伯格尔森博士组成的备受尊敬的内科科学家组成的科学咨询委员会。此外，保罗·奥菲特博士、莫里斯·希尔曼疫苗学捐赠主席、宾夕法尼亚大学儿科教授和CHOP传染病主席将帮助指导我的临床发展。这项研究的重点是不变自然杀伤T细胞(INKT)介导的肿瘤免疫监视。INKT细胞是先天淋巴细胞，在宿主免疫中发挥关键作用， 包括对特定病原体和肿瘤的保护。已知这些细胞通过大量产生细胞因子来间接参与抗肿瘤反应，这些细胞因子可以促进自然杀伤(NK)和CD8+T细胞的抗肿瘤活性。在使用iNKT细胞杂交瘤DN3A4-1.2以及原代小鼠和人iNKT细胞进行的研究中，我发现当靶细胞负载刺激糖脂抗原时，iNKT细胞本身在体外对CD1d阳性肿瘤显示出直接的细胞毒作用。此外，在没有穿孔素的情况下，这种体外iNKT细胞毒性大大降低。最后，在一个体内模型中，在没有其他淋巴细胞的情况下可以评估iNKT细胞的直接肿瘤监视能力，我发现小鼠iNKT细胞足以清除CD1d表达的糖脂负载的肿瘤。基于这些观察，我建议进一步剖析iNKT细胞介导的免疫监控的机制。具体地说，我将确定通过小鼠和人类iNKT细胞控制体内肿瘤生长的最佳要求，以及刺激糖脂抗原在这一过程中的重要性(目标1)。在AIM 2中，我将研究其表达和细胞定位 对静息和激活的小鼠和人iNKT细胞中的细胞溶解效应分子进行研究，然后评估这些分子对体内肿瘤免疫监视的需求。最后，在AIM 3中，我将检测刺激性细胞因子IL-2在体内外是否增强iNKT细胞的细胞毒作用，并探索iNKT细胞杀伤能力增强的机制。拟议研究的完成将有助于确定直接轴 在体内对iNKT细胞的细胞毒性和肿瘤免疫监测的研究，并将为这些细胞在癌症过继细胞治疗中的未来应用提供见解。总体而言，乔普和宾夕法尼亚大学为我作为内科科学家的培训提供了一个理想的科学环境。我将利用我的合作导师和科学咨询委员会成员的智力优势和学术记录，以及CHOP和宾夕法尼亚大学提供的强大的专业知识、设施和资源来完成这一拟议的培训计划。

DESCRIPTION (provided by applicant): This proposal describes a 4-year training program for the development of my academic career in Pediatric Infectious Diseases. I have completed formal residency in General Pediatrics and subspecialty fellowship training in Pediatric Infectious Diseases at the Children's Hospital of Philadelphia (CHOP), and am now expanding my scientific skills in NKT cell biology and tumor immunology, and developing my clinical skills in the infectious diseases of immunocompetent and immunocompromised hosts. Drs. Kim Nichols and Gary Koretzky will be mentoring my scientific development. Dr. Nichols, the Director of the Hereditary Cancer Disposition Program at CHOP, and Associate Professor of Pediatrics at the University of Pennsylvania (Penn), is an expert in NKT cell biology. Dr. Nichols has a growing track record of developing the careers of fellows, post-doctoral trainees, and junior faculty. To make my mentoring team as strong as possible, Dr. Nichols has partnered with Dr. Koretzky, Vice-Chair of Research and Chief Scientific Officer in the Department of Medicine at Penn, and the Director of the Signal Transduction Program in the Abramson Family Cancer Research Institute. Dr. Koretzky is an international authority on lymphocyte biology and T cell signal transduction with extensive experience in successfully mentoring numerous students, post-doctoral fellows, and junior faculty members. To further promote my scientific development, I have composed a Scientific Advisory Committee of highly-regarded physician-scientists consisting of Drs. Jordan Orange, Stephan Grupp, and Jeffrey Bergelson. Additionally, Dr. Paul Offit, Maurice Hilleman Endowed Chair in Vaccinology, Professor of Pediatrics at Penn, and Chair of Infectious Diseases at CHOP, will help guide me regarding my clinical development. The proposed research focuses on the tumor immunosurveillance mediated by invariant natural killer T (iNKT) cells. iNKT cells are innate lymphocytes that play critical roles in host immunity, including protection from specific pathogens and tumors. These cells are known to participate in anti-tumor responses indirectly via the robust production of cytokines that promote the anti-tumor activity of natural killer (NK) and CD8+ T cells. In studies using the iNKT cell hybridoma line, DN3A4-1.2, and primary murine and human iNKT cells, I find that iNKT cells themselves exhibit direct cytotoxicity in vitro against CD1d-positive tumors when the target cells are loaded with stimulatory glycolipid antigens. Additionally, in the absence of perforin, this in vitro iNKT cytotoxicity is greatly reduced. Finally, in an in vivo model in which the direct tumor surveillanc capacity of iNKT cells can be evaluated in the absence of other lymphocytes, I find that murine iNKT cells are sufficient for clearance of CD1d-expressing glycolipid-loaded tumors. Based on these observations, I propose to further dissect the mechanisms governing iNKT cell-mediated immunosurveillance. Specifically, I will define the optimal requirements for control of tumor growth in vivo by murine and human iNKT cells and the importance of stimulatory glycolipid antigens in this process (AIM 1). In AIM 2, I will examine the expression and cellular localization of cytolytic effector molecules in resting and activated murine and human iNKT cells, and then evaluate the requirement of these molecules for in vivo tumor immunosurveillance. Finally, in AIM 3, I will examine whether the stimulatory cytokine interleukin-2 augments iNKT cell cytotoxicity in vitro and in vivo and I will explore the mechanisms underlying the augmentation of iNKT cell killing capacity. The completion of the proposed studies will help define the direct axis of iNKT cell cytotoxicity and tumor immunosurveillance in vivo, and should provide insights into the future use of these cells in the adoptive cellular therapy of cancer. Collectively, CHOP and Penn provide an ideal scientific environment for my training as a physician-scientist. I will take advantage of the intellectual strength and academic track-record of my co- mentors and scientific advisory committee members, and the robust availability of expertise, facilities, and resources afforded at CHOP and Penn to accomplish this proposed training program.