BACKGROUND NR2F6 has been proposed as an alternative cancer immune checkpoint in the effector T cell compartment. However, a realistic assessment of the in vivo therapeutic potential of NR2F6 requires acute depletion. METHODS Employing primary T cells isolated from Cas9-transgenic mice for electroporation of chemically synthesized sgRNA, we established a CRISPR/Cas9-mediated acute knockout protocol of Nr2f6 in primary mouse T cells. RESULTS Analyzing these Nr2f6CRISPR/Cas9 knockout T cells, we reproducibly observed a hyper-reactive effector phenotype upon CD3/CD28 stimulation in vitro, highly reminiscent to Nr2f6-/- T cells. Importantly, CRISPR/Cas9-mediated Nr2f6 ablation prior to adoptive cell therapy (ACT) of autologous polyclonal T cells into wild-type tumor-bearing recipient mice in combination with PD-L1 or CTLA-4 tumor immune checkpoint blockade significantly delayed MC38 tumor progression and induced superior survival, thus further validating a T cell-inhibitory function of NR2F6 during tumor progression. CONCLUSIONS These findings indicate that Nr2f6CRISPR/Cas9 knockout T cells are comparable to germline Nr2f6-/- T cells, a result providing an independent confirmation of the immune checkpoint function of lymphatic NR2F6. Taken together, CRISPR/Cas9-mediated acute Nr2f6 gene ablation in primary mouse T cells prior to ACT appeared feasible for potentiating established PD-L1 and CTLA-4 blockade therapies, thereby pioneering NR2F6 inhibition as a sensitizing target for augmented tumor regression. Video abstract.

中文翻译：

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靶向 T 细胞中的孤儿核受体 NR2F6 为肿瘤免疫检查点治疗做好准备。

背景 NR2F6 已被提议作为效应 T 细胞区室中的替代癌症免疫检查点。然而，对 NR2F6 体内治疗潜力的现实评估需要急性消耗。方法利用从 Cas9 转基因小鼠中分离的原代 T 细胞对化学合成的 sgRNA 进行电穿孔，我们在原代小鼠 T 细胞中建立了 CRISPR/Cas9 介导的 Nr2f6 急性敲除方案。结果 通过分析这些 Nr2f6CRISPR/Cas9 敲除 T 细胞，我们在体外 CD3/CD28 刺激下可重复观察到高反应性效应器表型，这与 Nr2f6-/- T 细胞非常相似。重要的，CRISPR/Cas9 介导的 Nr2f6 在过继细胞疗法 (ACT) 前将自体多克隆 T 细胞消融到野生型荷瘤受体小鼠体内，并结合 PD-L1 或 CTLA-4 肿瘤免疫检查点阻断显着延迟 MC38 肿瘤进展并诱导更高的存活率，从而进一步验证了 NR2F6 在肿瘤进展过程中的 T 细胞抑制功能。结论 这些发现表明 Nr2f6CRISPR/Cas9 敲除 T 细胞与生殖系 Nr2f6-/- T 细胞相当，这一结果提供了对淋巴 NR2F6 免疫检查点功能的独立确认。总之，在 ACT 之前，CRISPR/Cas9 介导的原代小鼠 T 细胞中的急性 Nr2f6 基因消融对于加强已建立的 PD-L1 和 CTLA-4 阻断疗法似乎是可行的，从而开创性地将 NR2F6 抑制作为增强肿瘤消退的敏化目标。视频摘要。