肿瘤细胞治疗研究
英文原题：Sleeping Beauty mutagenesis identifies BACH2 and other regulators of CD8(+) T-cell exhaustion, persistence in vivo, and CAR-T cell function under tumor-associated chronic antigen stimulation.
使用 DiSBey 小鼠进行可控 SB 诱变，为功能性筛选可改善 T 细胞表型（这些表型对其作为疗法的应用至关重要）的基因提供了一个新平台。我们的发现强调了 BACH2 在慢性抗原刺激条件下增强工程化 T 细胞功能中的剂量依赖性作用。
增强T细胞功能的基因是改善癌症工程化T细胞疗法的有前景靶点。尽管大量CRISPR敲除筛选已鉴定出增强T细胞持久性的关键基因，但采用Sleeping Beauty（SB）插入诱变——该方法通过与内源基因生成融合转录本来诱导功能获得（GOF）和功能缺失（LOF）突变——可能揭示既往方法所忽略的其他关键因子。
我们构建了在原代T细胞中携带多西环素（Dox）诱导型SB诱变系统（DiSBey）的转基因小鼠。利用DiSBey，我们筛选了在慢性抗原暴露下增强T细胞持久性的遗传改变。具体而言，将来自Dox喂养的DiSBey小鼠的CD8+ T细胞在18天内反复进行抗CD3刺激，以模拟慢性抗原刺激。随后，我们分别使用增强特异性tagmentation测序和RNA测序，从持续存在的DiSBey CD8+ T细胞中鉴定了SB转座子基因组插入位点及相应的融合转录本。
在慢性刺激下，SB 诱变的 CD8+ T 细胞表现出改善的持久性并减少了终末耗竭表型。在六项独立筛选中，我们鉴定了 38 个基因，这些基因被 SB 转座子 T2/Onc2 反复靶向，并在慢性抗 CD3 刺激下差异表达。T2/Onc2 插入 Bach2 和 Elmo1 在基因组水平被反复鉴定到，并与新生转录本表达改变相关。Bach2 被认为是 T 细胞记忆形成和对慢性病毒感染诱导的耗竭抵抗的关键调节因子，但在用于癌症治疗的工程化 T 细胞中研究较少；研究发现 Bach2 可在体外对抗耗竭，并在 B16-Ova 肿瘤模型中增强体内肿瘤持久性。此外，我们表明异位 Bach2 表达水平影响工程化 T 细胞的分化谱系，因为低水平 Bach2 过表达保留了更多功能性祖细胞耗竭 T 细胞，并表现出改善的治疗疗效。最后，在人 CART19-28 细胞中，BACH2 过表达增强了慢性癌症刺激后的细胞毒性和肿瘤控制。
BACKGROUND: Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. METHODS: We developed transgenic mice carrying D oxycycline (Dox)- i nducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8 + T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 + T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. RESULTS: Under chronic stimulation, SB -mutagenized CD8 + T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28 cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. CONCLUSIONS: Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.
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