In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo. PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39– Tim3– Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39– Tim3– Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT.

在用于癌症治疗的过继性T细胞转移（ACT）的背景下，至关重要的是产生 体外 大量的肿瘤特异性CD8 T细胞具有很高的持久性 体内。PD-1，Tim3和CD39已被提议作为肿瘤特异性肿瘤浸润CD8 T淋巴细胞（CD8 TILs）的标记。但是，这些分子由缺乏增殖潜能的终末分化的耗尽CD8 T细胞（Tex）高度表达。因此，需要优化策略来分离具有高增殖潜力的肿瘤特异性TIL，例如Tcf1 +前体耗尽的T细胞（Tpe）体内ACT的持久性。在这里，我们旨在定义细胞表面标志物，以明确鉴定用于精确细胞分选的类型，从而从总TIL中增加肿瘤特异性PD-1 + Tcf1 + Tpe的纯度。对来自B16肿瘤和原发性人黑素瘤肿瘤的Tpe与Tex CD8 TIL亚组进行转录组学分析，结果表明Tpes富含猛击6 和缺乏 Entpd1 和 Havcr2表达，分别编码Slamf6，CD39和Tim3细胞表面蛋白。确实，我们通过流式细胞术观察到CD39–Tim3–Slamf6 + PD-1 +细胞在B16.OVA肿瘤中对肿瘤特异性PD-1 + Tcf1 + OT1 TILs产生最大富集。此外，与CD39 +对应物或大量PD-1 + TILs相比，该人群在急性感染召回反应后显示出更高的再扩张能力。因此，我们报告了Tpes的增强分类策略（CD39–Tim3–Slamf6 + PD-1 +）。总之，我们表明优化CD8 TIL细胞分选策略是提高召回能力和体内 ACT中转移的细胞的持久性。