Spatial genomic tools to interrogate T cell clonotypes, tumor clones and the microenvironment

当代免疫疗法的临床成功凸显了肿瘤浸润性细胞的关键作用。 在介导抗肿瘤免疫中的作用，并普遍将肿瘤内T细胞的存在与 治疗反应。然而，到目前为止，一种系统的方法来评估T细胞状态，它们的克隆 身份和本土化是相关的，一直不可能。近年来，吴实验室已经取得了几项成就 显著的技术进步，包括产生一种同类最佳的人类白细胞抗原I类表位预测器(HLAthena)；a 用于单细胞TCR测序的高度稳健的靶向平板方法(RhTCRseq)；以及一种用于 将数百个TCR的克隆并行化，以便可以询问它们以明确地将TCR与其 抗原特异性。因为肿瘤反应性T细胞和恶性克隆之间发生了动态相互作用 在定义的组织空间顺序中，我们有价值的新见解激励我们研究空间是如何 肿瘤特异性T细胞的组织与肿瘤克隆的原位定位有关。我们假设这一抗原 特异性，驱动T细胞和肿瘤细胞之间的相互作用，影响不同的区域定位 在基线和治疗背景下肿瘤微环境中T细胞的数量；相反，这一知识 的空间定位识别针对不同抗原类型的T细胞克隆。Slide-Seq技术，由 Chen实验室提供了一条易于处理且令人兴奋的途径，通过实现可扩展的 对人类和小鼠肿瘤组织进行深入分析的方法。通过扩展 这种无偏细胞分辨率空间捕获方法的能力，我们的目标是获得组织水平的理解 T细胞克隆的丰度和功能状态以及它们在肿瘤内的空间取向 微环境是相互联系的。特别是，这项研究将解决T细胞克隆的空间组织和 人和小鼠肿瘤中的肿瘤亚克隆。我们的技术目标将是提高 转录捕获技术，扩展了包括对肿瘤突变的可靠检测的能力，并 开发一套分析工具，以多模式方式整合转录、DNA水平和TCR水平 信息(目标1-2)。以肾细胞癌为例，我们将评估T细胞的细胞定位模式 TCR和DNA综合空间分析中克隆与肿瘤亚克隆和间质细胞的关系 幻灯片序列数据(目标2)。最后，我们将评估抗原特异性的影响(由Robust最终评估 本实验室建立的TCR重建和询问方法)对T细胞表型和定位的影响 TCR和DNA Slide-Seq整合了基线和免疫检查点阻断后的空间分析 新抗原疫苗。(目标3)总的来说，我们将开发和测试这些工具来理解空间局部化 驱动免疫反应的细胞网络，以及T细胞受体与肿瘤的关系 微环境。我们工作的完成将产生一个全面的工具集来使分子， 肿瘤免疫反应的细胞学和组织学理解。

The contemporary clinical successes of immunotherapies have highlighted the key role of tumor-infiltrating cells in mediating anti-tumor immunity and have generally associated the presence of T cells within tumors with therapeutic response. However, until now, a systematic approach for evaluating how T cell state, their clonal identity and localization are related has not been possible. In recent years, the Wu lab has achieved several notable technical advances, including generation of a best-in-class HLA class I epitope predictor (HLAthena); a highly robust targeted plate-based method for single-cell TCR sequencing (rhTCRseq); and a means to parallelize the cloning of hundreds of TCRs such that they can be interrogated to definitively link a TCR with its antigen specificity. Because dynamic interactions between tumor-reactive T cells and malignant clones occur within the defined spatial ordering of tissue, our valuable new insights motivate us to investigate how the spatial organization of tumor-specific T cells relates to in situ positioning of tumor clones. We hypothesize that antigen specificity, which drives the interactions between T cells and tumor cells, impacts the distinct regional localization of T cells within the tumor microenvironment at baseline and in the context of therapy; conversely, that knowledge of spatial localization identifies T cell clones specific for distinct antigen types. Slide-seq technology, created by the Chen lab, provides a tractable and exciting path to investigate this hypothesis by implementing a scalable approach to undertake in-depth analyses of informative human and murine tumor tissues. By expanding the capabilities of this unbiased cellular resolution spatial capture method, we aim to gain tissue level understanding of how the abundance and functional state of T cell clones and their spatial orientation within the tumor microenvironment are linked. In particular, the study will address the spatial organization of T cell clones and tumor subclones in human and mouse tumors. Our technology goals will be to increase the efficiency of transcript capture of the technology, extend the capability to include robust detection of tumor mutations, and to develop a suite of analytic tools to integrate in a multi model fashion the transcript, DNA-level and TCR levels of information (Aims 1-2). Focusing on RCC tumors, we will evaluate the cell-cell localization patterns of T cell clones in relation to tumor subclones and stromal cells through integrated spatial analyses of TCR and DNA Slide-seq data (Aim 2). Finally, we will evaluate the impact of antigen specificity (definitively assessed by robust TCR reconstruction and interrogation methods established in our lab) on T cell phenotype and localization using TCR and DNA Slide-seq integrated spatial analyses at baseline and following immune checkpoint blockade and neoantigen vaccine. (Aim 3) Altogether, we will develop and test these tools to understand spatially localized cellular networks which drive immune response, and T-cell receptor relationships with the tumor microenvironment. The completion of our work will yield a comprehensive toolset to enable a molecular, cellular and histological understanding of the tumor immune response.