Functional memory T cells are derived from exhausted clones and expanded by checkpoint blockade [scRNATCR_rechallenge]

Immune checkpoint blockade can facilitate tumor clearance by T cells, resulting in long term patient survival. However, the capacity of exhausted CD8+ T cells (Tex), present during chronic antigen exposure, to form memory after antigen clearance remains unclear. Here, we performed longitudinal single cell RNA/T cell receptor sequencing and ATAC-sequencing on antigen-specific T cells after the peripheral clearance of chronic lymphocytic choriomeningitis virus infection. These data revealed the formation of a robust population of memory CD8+ T cells that transcriptionally, epigenetically, and functionally resemble central memory T cells (Tcm) that form after clearance of acute infection. To lineage trace the origin and memory recall response of Tex-derived memory clones, we utilized T cell receptor sequencing over the course of primary infection and rechallenge. We show that chronic Tcm are a clonally distinct lineage of Tex derived from progenitor exhausted cells, persist long-term in the absence of antigen, and undergo rapid clonal expansion during rechallenge. Finally, we demonstrate that αPD-L1 immune checkpoint blockade selectively expands clones which form Tcm after clearance. Together, these data support the concept that chronically stimulated T cells form bona fide functional memory T cells through an analogous differentiation pathway to acutely stimulated T cells, which may have significant implications for enhancing immune memory to cancer through checkpoint blockade and vaccination. To track T cell clones during rechallenge, CD8+ T cells were isolated from the spleens of mice that cleared LCMV-Cl13 or LCMV-Arm infection. Half of the cells were used for bulk TCR sequencing prior to rechallenge. The remaining cells were transferred into new host mice, challenged with LCMV-Arm. At 8 days post-infection transferred T cells were sorted, and scRNA/TCR-seq was performed. Pre-rechallenge TCR-seq can be found at doi.org/10.5281/zenodo.14648171

免疫检查点阻断（Immune checkpoint blockade）可促进T细胞介导的肿瘤清除，进而实现患者长期生存。然而，在慢性抗原暴露过程中存在的耗竭型CD8+ T细胞（Tex），其在抗原清除后形成记忆细胞的能力仍未明确。
本研究针对慢性淋巴细胞性脉络丛脑膜炎病毒（Lymphocytic choriomeningitis virus, LCMV）慢性感染外周清除后的抗原特异性T细胞，开展了纵向单细胞RNA/T细胞受体测序及ATAC测序（ATAC-sequencing）。上述数据揭示了一类稳定的记忆性CD8+ T细胞群的形成，该细胞群在转录组、表观基因组及功能层面均与急性感染清除后形成的中枢记忆性T细胞（central memory T cells, Tcm）高度相似。
为对Tex来源的记忆克隆的起源及记忆再次应答进行谱系示踪，本研究在原发性感染及再次感染过程中开展了T细胞受体测序。研究显示，慢性感染相关中枢记忆性T细胞（chronic Tcm）是一类克隆谱系独特的Tex亚群，其起源于前体耗竭细胞，在无抗原存在的情况下可长期存活，并在再次感染过程中发生快速克隆扩增。
最后，本研究证实，αPD-L1免疫检查点阻断可选择性扩增那些在感染清除后可形成Tcm的克隆。
综上，本研究数据支持以下观点：慢性刺激的T细胞可通过与急性刺激T细胞相似的分化途径，形成真正具有功能活性的记忆性T细胞，这一发现或可为通过免疫检查点阻断及疫苗接种增强抗肿瘤免疫记忆提供重要理论参考。
为在再次感染过程中追踪T细胞克隆，本研究从清除了LCMV-Cl13或LCMV-Arm感染的小鼠脾脏中分离CD8+ T细胞。
将一半细胞在再次感染前用于批量T细胞受体测序；剩余细胞被转移至新的受体小鼠体内，并以LCMV-Arm进行感染攻击。
在感染后第8天，对转移的T细胞进行分选，并开展单细胞RNA/T细胞受体测序。
再次感染前的T细胞受体测序数据可在doi.org/10.5281/zenodo.14648171获取。