B cell directed CAR-T cell therapy results in activation of CD8+ cytotoxic CAR-negative bystander T cells in both non-human primates and patients

There is growing appreciation for the emergence of CARneg bystander T cells after CAR-T cell infusion. However, their phenotypic and transcriptomic hallmarks and mechanisms of activation remain uncertain. We performed single-cell RNA-Seq (scRNA-Seq) on non-human primate (NHP) and patient-derived T cells to interrogate CARneg T cells following B cell targeted CAR-T cell therapy. In a NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR-T cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of NK-cell markers (KIR3DL2, CD160, KLRD1), chemokines and chemokine receptors (CCL5, XCL1, CCR9), and downregulation of naive T cell-associated genes (SELL, CD28). A transcriptionally similar population was identified in patients following Tisangelecleucel infusion. Mechanistic studies revealed that IL-2 and IL-15 exposure induced bystander-like CD8+ T cells. These T cells efficiently killed leukemic cells through a TCR-independent mechanism. Together, these data identify bystander CD8+ T cells as a novel mechanism by which CAR-T cell infusion can induce further anti-leukemic activity, measurable in both NHP and in patients. scRNA-Seq of peripheral blood T cells obtained from NHPs undergoing CAR-T treatment

目前，学界对嵌合抗原受体T（CAR-T）细胞输注后CAR阴性（CARneg）旁观者T细胞的出现愈发重视，但其表型、转录组特征及激活机制仍未明确。本研究对非人灵长类动物（NHP）及患者来源的T细胞开展单细胞RNA测序（scRNA-Seq），以探究靶向B细胞的CAR-T细胞疗法后CAR阴性T细胞的特征。在非人灵长类动物模型中，我们观察到CAR-T细胞扩增阶段出现了一群独特的活化CD8+ CAR阴性T细胞。这类旁观者CD8+ CAR阴性T细胞具有独特的转录特征：上调自然杀伤细胞（NK细胞）标志物（KIR3DL2、CD160、KLRD1）、趋化因子及趋化因子受体（CCL5、XCL1、CCR9），同时下调初始T细胞相关基因（SELL、CD28）。在替沙仑赛（Tisangelecleucel）输注后的患者体内，同样检测到了转录特征相似的细胞亚群。机制研究显示，白细胞介素2（IL-2）与白细胞介素15（IL-15）的暴露可诱导产生类旁观者CD8+ T细胞，这类细胞可通过T细胞受体（TCR）非依赖机制高效杀伤白血病细胞。综上，本研究证实旁观者CD8+ T细胞是CAR-T细胞输注可诱导额外抗白血病活性的新机制，该现象在非人灵长类动物及患者体内均已得到验证。本研究的数据集为对接受CAR-T治疗的非人灵长类动物的外周血T细胞开展的单细胞RNA测序。