T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen–specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma—combined with transgenic reexpression of identified TCRs by CRISPR-Cas9–mediated orthotopic TCR replacement—revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

中文翻译：

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最近激活的特征确定了一个循环 T 细胞区室，其中包含具有高亲和力的肿瘤特异性抗原受体

T 细胞受体 (TCR) 亲和力被认为是肿瘤特异性 T 细胞时空命运和保护能力的主要决定因素。然而，在空间和时间上用已知的 TCR 亲和力在体内监测多克隆 T 细胞反应仍然具有挑战性。在这里，我们在一个完善的还原论临床前肿瘤模型和人类黑色素瘤患者中研究了具有不同亲合力的 TCR 的肿瘤新抗原特异性 T 细胞的命运和功能。为此，我们在接种了 MC38 OVA 肿瘤的小鼠中使用了具有深入表征的 TCR 的多克隆 T 细胞转移以及流式细胞术表型分析。从具有高亲和力的 TCR 的逆转录小鼠中转移 T 细胞可产生最佳的肿瘤保护。不料，我们发现肿瘤内的高亲和力和低亲和力 T 细胞同样丰富，并采用一致的衰竭表型迹象。在肿瘤外，高亲和力 TCR T 细胞通常不会过多，而是选择性富集在具有中间 PD-1 蛋白表达的 T 细胞群中。对两名黑色素瘤患者的新抗原特异性 T 细胞进行单细胞测序——结合通过 CRISPR-Cas9 介导的原位 TCR 替代对已识别 TCR 进行转基因再表达——揭示了高功能性 TCR 在具有最近激活的 RNA 特征的 T 细胞中富集. 此外，在 130 种表面蛋白候选物中，PD-1 表面表达最一致地富含功能性 TCR。一起，