输注产品中干细胞样记忆 T 细胞的高频率与多项 T 细胞治疗试验中的优异患者结果相关。在此，我们分析了已发表的 CRISPR 激活筛选，以确定可用于增强 CD8 T 细胞中干细胞样行为的转录调节因子。使用 IFN-γ 的产生作为 CD8 T 细胞终末分化的代表，LMO4 成为抑制效应细胞发育的热门药物之一。一致地，我们发现 Lmo4 在 CD8 T 细胞激活时下调，但在促进干样 T 细胞形成的培养条件下保持不变。通过采用合成生物学方法在抗肿瘤 CD8 T 细胞中异位表达 LMO4，我们能够选择性扩增并增强转导细胞的持久性，同时限制其终末分化和衰老。 LMO4过表达促进了调节干细胞性的转录程序，增加了干细胞样CD8记忆T细胞的数量并增强了它们的多功能性和记忆能力。在同基因和异种移植肿瘤模型中进行测试时，LMO4 过表达增强了 CD8 T 细胞抗肿瘤免疫力，从而增强了肿瘤消退。 LMO4 不是直接调节基因转录，而是与 JAK1 结合，并增强 STAT3 信号传导以响应 IL-21，诱导对记忆反应至关重要的靶基因（Tcf7、Socs3、Junb 和 Zfp36）的表达。 Stat3 的 CRISPR/Cas9 删除使 LMO4 赋予的增强记忆特征无效，从而消除了 LMO4 过表达的治疗益处。这些结果确立了 LMO4 过表达作为增强 CD8 T 细胞干性的有效策略，提供了一种新的合成生物学工具来增强基于 T 细胞的免疫疗法的功效。© 2024。作者。

High frequencies of stem-like memory T cells in infusion products correlate with superior patient outcomes across multiple T cell therapy trials. Herein, we analyzed a published CRISPR activation screening to identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8+ T cells. Using IFN-γ production as a proxy for CD8+ T cell terminal differentiation, LMO4 emerged among the top hits inhibiting the development of effectors cells. Consistently, we found that Lmo4 was downregulated upon CD8+ T cell activation but maintained under culture conditions facilitating the formation of stem-like T cells. By employing a synthetic biology approach to ectopically express LMO4 in antitumor CD8+ T cells, we enabled selective expansion and enhanced persistence of transduced cells, while limiting their terminal differentiation and senescence. LMO4 overexpression promoted transcriptional programs regulating stemness, increasing the numbers of stem-like CD8+ memory T cells and enhancing their polyfunctionality and recall capacity. When tested in syngeneic and xenograft tumor models, LMO4 overexpression boosted CD8+ T cell antitumor immunity, resulting in enhanced tumor regression. Rather than directly modulating gene transcription, LMO4 bound to JAK1 and potentiated STAT3 signaling in response to IL-21, inducing the expression of target genes (Tcf7, Socs3, Junb, and Zfp36) crucial for memory responses. CRISPR/Cas9-deletion of Stat3 nullified the enhanced memory signature conferred by LMO4, thereby abrogating the therapeutic benefit of LMO4 overexpression. These results establish LMO4 overexpression as an effective strategy to boost CD8+ T cell stemness, providing a new synthetic biology tool to bolster the efficacy of T cell-based immunotherapies.© 2024. The Author(s).