Fluorescence molecular tomography to study T cell infiltration into tumors

描述（由申请人提供）：肿瘤中CD 8 T细胞（TCD 8）的存在是患者生存的阳性指标。同样清楚的是，临床上对新一代免疫疗法有反应的患者是那些在治疗前免疫肿瘤浸润已经明显的患者。另一方面，浸润肿瘤的过继转移的肿瘤特异性效应物的分数令人惊讶地小，这需要在细胞内进行细胞内转移。 大量T细胞的体外扩增结合淋巴细胞清除以支持体内进一步扩增。因此，T细胞的有限浸润是肿瘤控制的主要障碍，超过免疫抑制的障碍。T细胞上的归巢受体（Homing receptors，HR）及其相应的血管配体是TCD 8效应子浸润外周组织所必需的。然而，我们缺乏对哪些HR控制向肿瘤的迁移、这如何受到肿瘤血管系统上相应配体表达的影响以及这些相同HR也能够浸润到正常组织中的程度（可能与肿瘤浸润竞争）的充分理解。此外，TCD 8效应物在外渗后的滞留可通过其识别肿瘤细胞或肿瘤内Ag呈递细胞上的Ag的能力或通过滞留整合素的作用来确定。大多数肿瘤内T细胞的研究依赖于 流式细胞术、组织学和阻断或敲除方法，以在单个时间点研究这些问题中的一些。在这里，我们建议使用一种新的成像技术，荧光分子断层扫描（FMT），纵向监测TCD 8在荷瘤动物在真实的时间。FMT仅在一项初步研究中用于体内观察TCD 8。因此，该应用程序的某些方面旨在优化FMT的使用，期望它将得到广泛使用。该应用程序的更基本的方面将提供更好的理解T细胞进入和退出肿瘤和其他外周组织的运输，分布和动力学; T细胞上的归巢受体（HR），其相应的血管配体和抗原在促进肿瘤进入和保留中的重要性;以及肿瘤微环境在控制这些过程中的作用。该应用程序是两名研究人员之间的合作，他们在肿瘤免疫学和肿瘤血管生物学方面具有互补的知识专长，并通过多色流式细胞术和先进的整体动物成像技术在细胞免疫学/单细胞分析方面具有技术专长。本申请的具体目的是：1）建立使用FMT长期测量T细胞分布和运输的系统; 2）研究荷瘤小鼠中TCD 8效应物的分布和动力学;和3）建立炎症、内源性TCD 8效应物和Treg在控制TCD 8浸润到肿瘤中的作用和肿瘤相关脉管系统的性质。我们希望本申请中提出的工作结果将支持未来的多研究者R 01或P01项目申请，其中将进行此类综合研究。

DESCRIPTION (provided by applicant): The presence of CD8 T cell (TCD8) in tumors is a positive indicator of patient survival. It has also become clear that patients who respond clinically to new generation immunotherapies are those in which an immunological tumor infiltrate is already evident prior to treatment. On the other hand, the fraction of adoptively transferred tumor-specific effectors that infiltrate tumors is surprisingly small, necessitating in vitro expansion of large numbers of T cells in conjunction with lymphodepletion to support further expansion in vivo. Thus, limited infiltration of T cells is a major barrier to tumor contro over and above the hurdle of immunosuppression. This is an application to understand the basis for this limited infiltration and seek ways to overcome it. Homing receptors (HR) on T cells and their corresponding vascular ligands are essential for TCD8 effectors to infiltrate peripheral tissues. However, we lack a full understanding of which HR control migration into tumors, how this is influenced by expression of the corresponding ligands on tumor vasculature, and the extent to which these same HR also enable infiltration into normal tissues, potentially in competition with tumor infiltration. In addition, the retention of TCD8 effectors after extravasatin may be determined by their ability to recognize Ag on tumor cells or intratumoral Ag presenting cells, or by the action of retention integrins. Most studies of intratumoral T cells have relied on flow cytometry, histology, and blockade or knockout approaches to investigate some of these issues at single time points. Here, we propose to use a new imaging technique, Fluorescence Molecular Tomography (FMT), to longitudinally monitor TCD8 in tumor bearing animals in real time. FMT has been used to visualize TCD8 in vivo in only a single preliminary study. Thus, some aspects of this application are designed to optimize the use of FMT, with the expectation that it will become widely used. The more fundamental aspects of the application will provide greater understanding of the trafficking, distribution and dynamics of T cell entry and exit into tumors and other peripheral tissues; the importance of homing receptors (HR) on T cells, their corresponding vascular ligands, and antigen in promoting tumor entry and retention; and the role of the tumor microenvironment in controlling these processes. This application is a collaboration between two investigators with complementary intellectual expertise in tumor immunology and tumor vascular biology and technical expertise in cellular immunology / single cell analysis by multi-color flow cytometry and advanced whole animal imaging techniques. The specific aims of this application are: 1) To establish a system for long-term measurements of T cell distribution and trafficking using FMT; 2) To investigate the distribution and dynamics of TCD8 effectors in tumor bearing mice; and 3) To establish the role of inflammation, endogenous TCD8 effectors, and Treg in controlling TCD8 infiltration into tumors and the properties of the tumor- associated vasculature. We expect the results from the work proposed in this application will support a future multi-investigator R01 or P01 project application in which such comprehensive studies will be undertaken.