PO.CL05.13 · 临床研究
通过单细胞转录组分析鉴定T细胞反应性的新型细胞表面标志物
Identifying new cell surface markers of T-cell reactivity through single cell transcriptomic analysis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：在一项2期试验中，经筛选的肿瘤浸润淋巴细胞（SEL-TIL）联合帕博利珠单抗的过继转移已取得23.5%的临床缓解率。1 在IL-2中扩增后，通过体外反应性检测选择用于治疗的TIL培养物，即将TIL与候选自体肿瘤靶点（多肽、串联微基因（TMG）和自体肿瘤类器官）共培养。目前对新抗原反应性的检测依赖于4-1BB、OX40和干扰素-γ（IFN-gamma）的上调。2 临床上给予更多新抗原反应性CD4+ TIL与缓解改善相关。1 然而，可能还存在其他反应性标志物，能够检测新抗原相关片段，并在CD4+和CD8+ T细胞中产生更多样化的最终治疗产品。
方法：新抗原反应性TIL的初步筛选和选择采用标准筛选方法，通过流式细胞术检测4-1BB和OX40的上调，以及通过ELISpot检测IFN-gamma。随后选择这些具有已知反应性的样本进行转录组学分析。使用10x Genomics Chromium平台，对已知的针对类器官的肿瘤反应性片段及阴性对照进行scRNA测序。通过TCR的条形码哈希标记来追踪各条件。同时还使用了CITE-Seq和TCR-seq。使用Seurat完成聚类和分析。
结果：在单个细胞簇中发现TNFRSF9（4-1BB）和IFNG（IFN-gamma）的上调。此外还发现一个更大的细胞簇，表达包括TNFRSF18（GITR）在内的其他基因。在阴性对照条件下未观察到GITR及其他标志物的这种激活。在其他验证样本中，作为CD8 T细胞针对自体类器官激活的细胞表面标志物进行检测时，通过流式细胞术成功观察到GITR，其水平等于或高于4-1BB。
结论：使用scRNA转录组分析的方法是鉴定新型上调标志物的一种成功且可行的方法。GITR是CD8+ T细胞肿瘤反应性的一个潜在标志物，但仍需在更多样本中进一步验证并评估其抗肿瘤效应。
查看英文原文 English abstract
Background : Adoptive transfer of selected tumor infiltrating lymphocytes (SEL-TIL)+ pembrolizumab has yielded clinical responses of 23.5% in a phases 2 trial. 1 Following expansion in IL-2, TIL cultures are selected for treatment based on in vitro reactivity assays in which TIL are co-cultured with candidate autologous tumor targets, (peptides, tandem minigenes (TMGs), and autologous tumor organoid). Detection of neoantigen reactivity currently relies on 4-1BB, OX40, and interferon-gamma (IFN-gamma) upregulation. 2 Clinical administration of more neoantigen reactive CD4+ TIL is associated with improved responses. 1 However, there may be additional reactivity markers that would allow for detection of neoantigen relevant fragments and more diverse final treatment products in both CD4+ and CD8+ T-cells.
Methods : Initial screening and selection of neoantigen reactive TIL completed using standard screening via upregulation of 4-1BB, and OX40 by flowcytometry and IFN-gamma by ELISpot. These samples with known reactivity were then selected for transcriptomics. scRNA sequencing was performed using 10x Genomics Chromium platform on known tumor-reactive fragments to organoid and negative controls. Conditions were tracked with barcode hashing of TCRs. CITE-Seq, and TCR-seq were also used. Clustering and analysis completed using Seurat.
Results : Upregulation of TNFRSF9 (4-1BB) and IFNG (IFN-gamma) were found in a single cluster. An additional larger cluster was found expressing additional genes including TNFRSF18 (GITR). This activation of GITR, and other markers was not seen in negative control conditions. In additional validation samples, GITR was successfully observed by flow cytometry equal to or greater than 4-1BB when tested as a cell surface marker of activation for CD8 T-cells against autologous organoid.
Conclusions : The methodology of using scRNA transcriptomic analysis is a successful and viable method for identifying new markers of upregulation. GITR is a potential marker of CD8+ T-cell tumor reactivity, however, it requires further validation in additional samples and evaluation of antitumor effects.
利益披露 Disclosure
A. A. Hakim, None..
I. S. Goldlust, None..
A. Bhasin, None..
B. Bui, None..
N. D. Klemen, None..
F. J. Lowery, None.