The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4+ helper T (TH) cells and CD8+ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of TH1 and TH2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

中文翻译：

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CAR-T 细胞激活的单细胞分析揭示了一种独立于分化的混合 TH1/TH2 反应。

嵌合抗原受体 (CAR) 工程 T 细胞的激活机制可能与携带天然 T 细胞受体的 T 细胞有很大不同，但这种差异仍然知之甚少。我们首次全面描述了抗 CD19/4-1BB/CD28/CD3ζ CAR-T 细胞在抗原特异性刺激下的单细胞水平转录和细胞因子特征。CD4+ 辅助性 T (TH) 细胞和 CD8+ 细胞毒性 CAR-T 细胞在直接杀死靶肿瘤细胞方面同样有效，并且它们的细胞毒性活性与一系列 TH1 和 TH2 标志性细胞因子的升高有关，例如干扰素 γ，肿瘤坏死因子 α、白细胞介素 5 (IL5) 和 IL13，如主转录因子基因 TBX21 和 GATA3 的表达所证实。然而，不是符合严格的 TH1 或 TH2 亚型，单细胞分析表明，主要反应是同一细胞中高度混合的 TH1/TH2 功能。调节性 T 细胞活性虽然在一小部分活化细胞中观察到，但出现在该混合 TH1/TH2 群体中。无论极化状态如何，大多数细胞都会产生粒细胞-巨噬细胞集落刺激因子 (GM-CSF)，进一步将 CAR-T 与经典 T 细胞进行对比。令人惊讶的是，细胞因子反应与分化状态的相关性极小，尽管检测到所有主要的分化子集，如幼稚、中央记忆、效应记忆和效应子。所有这些都表明 CAR 改造的 T 细胞的激活是一个典型的过程，它导致将 1 型和 2 型细胞因子与 GM-CSF 结合在一起的高度混合反应，支持多功能 CAR-T 细胞与临床试验中患者的客观反应相关的观点。这项工作提供了对 CAR 激活机制的新见解，并暗示了细胞功能测定的必要性，以表征 CAR-T 输注产品的质量并监测患者的治疗反应。