Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

原位鉴定工程化 T 细胞对于了解过继性 T 细胞治疗后患者体内这些细胞的位置、持久性和表型非常重要。虽然工程细胞通常通过流式细胞术在患者的新鲜组织或血液中进行表征，但很难将它们与福尔马林固定石蜡包埋 (FFPE) 组织活检中的内源细胞区分开来。为了克服这一限制，我们开发了一种方法，利用原位杂交 (ISH) 与土拨鼠肝炎转录后调节元件 (WPRE) 表征固定组织中的工程化 T 细胞，该元件常见于许多用于转导嵌合抗原受体 T 的慢病毒载体中。 CAR-T）和 T 细胞受体 T (TCR-T) 细胞，结合替代透化条件，允许在同一图像内进行后续多重免疫组织化学 (mIHC) 染色。这种新方法提供了通过 ISH 标记细胞的能力，同时对细胞相关蛋白进行染色，以对肿瘤内 CAR/TCR 修饰的 T 细胞进行免疫表型分析，并评估这些细胞在靶向/肿瘤外毒性中的潜在作用在其他组织中。