Combining targeting of the cancer-metabolome with cancer-associated stress antigens impacts transcriptomic heterogeneity, dynamics, and efficacy of engineered T-cells

The majority of cancers cannot be efficiently targeted with engineered T cell strategies. In this line, we explored how and whether ?dTCR-mediated cancer metabolome targeting can be combined with the attack of cancer-associated stress antigens, like NKG2D-ligands, in order to overcome limited targeting of many tumors, and at the same time skew heterogeneous transcriptomic signatures of engineered immune cells towards more active, and less exhausted phenotypes.Single cell transcriptomic analysis revealed that only NKG2D-4-1BBCD28TM chimera reprogrammed TEGs significantly, by skewing CD4+ TEG heterogeneity towards more adhesive, proliferative, cytotoxic, and less exhausted transcriptional signatures. Overall design: TEGs (T cells engineered with defined gamma delta TCRs), 'CD28 chimeras' (TEGs expressing the NKG2D-CD28wt chimera), '41BB chimeras' (TEGs expressing the NKG2D-4-1BBCD28TM chimera), '103_41BB chimeras' (TEGs expressing the 103-4-1BB chimera) were co-cultured together with RPMI 8226 cells in the presence of 10 µM PAM during 48h. The '_t' label marks samples that originated from a shared donor. After incubation, cells were sorted by expression of ?dTCR+CD4+ or ?dTCR+CD8+ (pan-?dTCR-PE, clone IMMU510; CD4-PB, clone RPA-T4; CD8-PerCP Cy5.5, RPA-T8) into 384-well PCR plates using ARIA II. Cells were sequenced according to the SORT-seq method based on CEL-Seq2, and libraries were sequenced with paired end sequencing on Nextseq 500.

绝大多数癌症无法通过工程化T细胞策略实现有效靶向。基于此研究方向，本工作探究了γδT细胞受体（γδ T Cell Receptor, γδTCR）介导的癌症代谢组靶向策略，能否与NKG2D配体（NKG2D ligand）等肿瘤相关应激抗原的杀伤作用相结合，以突破多数肿瘤的靶向限制难题；同时还探索了如何将工程化免疫细胞的异质性转录组特征重塑为更具活性、耗竭程度更低的表型。
单细胞转录组分析结果显示，仅NKG2D-4-1BBCD28TM嵌合受体可显著重编程TEGs（经工程化改造以表达特定γδTCR的T细胞，T cells engineered with defined gamma delta TCRs），将CD4+ TEGs的异质性转录组特征向更具黏附性、增殖性、细胞毒性且耗竭程度更低的方向倾斜。
实验整体设计：将TEGs（经工程化改造以表达特定γδTCR的T细胞，T cells engineered with defined gamma delta TCRs）、「CD28嵌合体」（表达NKG2D-CD28wt嵌合受体的TEGs）、「41BB嵌合体」（表达NKG2D-4-1BBCD28TM嵌合受体的TEGs）以及「103_41BB嵌合体」（表达103-4-1BB嵌合受体的TEGs）与RPMI 8226细胞系共培养，体系中添加10 μM PAM，孵育48小时。样本后缀「_t」表示其来自同一供体。孵育结束后，采用ARIA II流式分选仪，依据细胞表面标志物表达（PE标记的泛γδTCR抗体，克隆号IMMU510；PB标记的CD4抗体，克隆号RPA-T4；PerCP Cy5.5标记的CD8抗体，克隆号RPA-T8），将γδTCR+CD4+或γδTCR+CD8+细胞分选至384孔PCR板中。随后采用基于CEL-Seq2的SORT-seq技术对细胞进行建库，并使用Nextseq 500测序平台完成双端测序。