The importance of CD4+ tumor-infiltrating lymphocytes (TIL) in adoptive cell transfer

项目摘要/摘要 肿瘤浸润性淋巴细胞(TIL)的过继细胞转移(ACT)是一种有前途的治疗方案 在转移性黑色素瘤的临床试验中，癌症患者的客观应答率为30%-55% 病人。目前的TIL产生策略是以CD8+T细胞为中心的，尽管有迹象表明CD4+T细胞 在多种恶性肿瘤中流行，本质上是多功能的，并在 大量的临床和临床前应用。这项提案的总体目标是系统地 探讨CD4+TIL在ACT和免疫治疗耐药中的作用。我们假设 CD4+TIL有助于ACT的有效性，并提供了一种有效的机制来克服 对当前免疫治疗策略的抵抗力。为了研究这一假设，我们将1)确定 人黑色素瘤标本中CD4+TIL的功能和表型，2)阐明其作用机制 体内CD4+TIL抗肿瘤反应；3)检测CD4+TIL在克服耐药中的作用 去接受免疫治疗。在目标1中，这项研究将确定和验证CD4+TIL的内在决定因素 通过使用体外免疫学分析和生物信息学分析提供临床疗效 未识别的样本来自接受TIL的ACT患者。目标2将探讨潜在的机制 通过使用小鼠模型来解决未回答的抗原特异性和 由CD4+T细胞识别抗原。最后，目标3将利用复杂的小鼠和人类系统，包括 CRISPR/Cas9，探讨CD4+TIL在免疫治疗中的应用。这样做的结果 研究将为在ACT的TIL产品中包括CD4+T细胞提供理论依据，特别是在 免疫治疗耐药。通过这些目标的完成，麦克莱恩将经历一场密集的 生物信息学和分子生物学方面的高级免疫学和基本原理培训 为他成为一名成功的独立调查员做好准备。这次培训得到了他的全力支持 导师，莎莉·皮隆-托马斯博士，合作者，导师委员会和该机构的阿莫德·萨尔奈克博士， 莫菲特癌症中心和研究所。该培训计划的目标是促进职业发展。 麦克莱恩在基础和翻译研究方面的发展，通过1)肿瘤免疫学和 小鼠模型，2)数据分析和解释，3)口头陈述技能，4)科学写作技能，5) 参加科学会议。Pilon-Thomas博士和Sarnaik博士将每周与MacLean会面，提供 指导实验设计，并讨论数据分析和解释。麦克莱恩还将会见 他的指导委员会成员每季度一次，为他的进展提供了宝贵的补充 项目。麦克莱恩将积极参加免疫学高级课程，参加国家会议和 资助写作工作坊，并准备手稿，总体目标是从博士生过渡到 博士后研究员，最终成为翻译学术研究的独立研究员。

Project Summary/Abstract Adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) is a promising therapeutic option for cancer patients, demonstrating a 30-55% objective response rate in clinical trials with metastatic melanoma patients. Current strategies for TIL production are CD8+ T cell-centric, despite indications that CD4+ T cells are prevalent across multiple malignancies, are polyfunctional in nature, and provide therapeutic benefit in a multitude of clinical and pre-clinical applications. The overall objective of this proposal is to systematically investigate the role of CD4+ TIL in the settings of ACT and immunotherapeutic resistance. We hypothesize that CD4+ TIL are instrumental to the effectiveness of ACT and provide an effective mechanism to overcome resistance to current immunotherapy strategies. To investigate this hypothesis, we will 1) Determine the function and phenotype of CD4+ TIL from human melanoma samples, 2) Elucidate the mechanisms of the CD4+ TIL anti-tumor response in vivo, and 3) Examine the contribution of CD4+ TIL in overcoming resistance to immunotherapy. In Aim 1, this study will determine and validate the intrinsic determinants of CD4+ TIL that provide clinical efficacy through the use of in vitro immunologic assays and bioinformatics analysis based on de-identified samples from patients who received ACT with TIL. Aim 2 will approach the mechanism underlying in vivo efficacy through the use of mouse models to address unanswered questions of antigen specificity and antigen recognition by CD4+ T cells. Finally, Aim 3 will utilize complex murine and human systems, including CRISPR/Cas9, to investigate the use of CD4+ TIL to rescue resistance to immunotherapy. The results of this study will provide rationale for the inclusion of CD4+ T cells in TIL products for ACT, especially in the setting of immunotherapeutic resistance. Through the completion of these aims, MacLean will undergo an intensive training in advanced immunology and fundamental principles in bioinformatics and molecular biology that will prepare him for a successful career as an independent investigator. This training is fully supported by his mentor, Dr. Shari Pilon-Thomas, collaborator, Dr. Amod Sarnaik, mentorship committee and the institution, Moffitt Cancer Center and Research Institute. The objective of this training program is to foster the career development of MacLean in basic and translational research through mentorship in 1) tumor immunology and murine models, 2) data analysis and interpretation, 3) oral presentation skills, 4) scientific writing skills, and 5) participation in scientific meetings. Drs. Pilon-Thomas and Sarnaik will meet with MacLean weekly to provide guidance on experimental design and to discuss data analysis and interpretation. MacLean will also meet with members of his mentorship committee on a quarterly basis for valuable added insight into the progress of his project. MacLean will actively participate in advanced courses in immunology, attend national conferences and grant writing workshops, and prepare manuscripts with the overall goal of transitioning from Ph.D. student to postdoctoral fellow and ultimately an independent investigator in translational academic research.