Single-cell analysis of the dLN-tumor OL clones following anti-CD4 mAb treatment

The repertoire of tumor-infiltrating T cells is a novel perspective to characterize effective anti-tumor T cell responses. Previous studies reported that the oligoclonal expansion in tumor T-cell repertoire is associated with anti-tumor effects. However, the contribution of the expansion of diverse T-cell clones remained unclear. We demonstrated that the polyclonal fraction of tumor-reactive T-cell repertoire consisting of relatively minor clones, increased in tumor-bearing mice treated with anti-PD-L1 or anti-CD4 monoclonal antibodies (mAbs), while the oligoclonal fraction consisting of major clones was unchanged. Moreover, the polyclonal fraction was enriched with progenitor exhausted T cells, essential for a durable anti-tumor response, and more dependent on CCR7+ migratory dendritic cells, responsible for priming tumor-reactive T cells in the tumor-draining lymph node (dLN). These results proposed that the expansion of diverse tumor-reactive clones, “clonal spreading,” is an important mechanism by which anti-PD-L1 and anti-CD4 treatments exert robust and durable anti-tumor T-cell responses. Overall design: CD8+ TILs and CD8+ CD44high T cells in dLN were collected from a B16F10 tumor-bearing mouse treated with anti-CD4 mAb. Single-cell targeted RNA sequencing combined with TCR sequencing (scRNA/TCRseq) was perfomed on CD8+ TILs. Bulk TCR sequencing was performed on dLN CD8+ CD44high T cells. T cell clones that overlap between tumor and dLN were identified.

肿瘤浸润T细胞（tumor-infiltrating T cells）的受体库是表征有效抗肿瘤T细胞应答的全新研究视角。既往研究显示，肿瘤T细胞受体库中的寡克隆扩增与抗肿瘤效应存在关联，但多样化T细胞克隆扩增的具体贡献仍未明确。本研究证实，经抗PD-L1或抗CD4单克隆抗体（monoclonal antibodies, mAbs）处理的荷瘤小鼠体内，由相对次要克隆构成的肿瘤反应性T细胞受体库多克隆组分比例显著升高，而由优势克隆构成的寡克隆组分比例未发生明显变化。此外，该多克隆组分富含祖细胞耗竭T细胞——这类细胞对持久抗肿瘤应答至关重要，且其形成更依赖于CCR7阳性迁移性树突状细胞，后者负责在肿瘤引流淋巴结（tumor-draining lymph node, dLN）中启动肿瘤反应性T细胞的致敏过程。上述结果表明，多样化肿瘤反应性克隆的扩增——即“克隆扩散（clonal spreading）”——是抗PD-L1与抗CD4治疗能够发挥强效且持久抗肿瘤T细胞应答的重要机制。实验整体设计：从经抗CD4单克隆抗体处理的B16F10荷瘤小鼠体内，采集肿瘤引流淋巴结中的CD8阳性肿瘤浸润T细胞（CD8+ TILs）与CD8阳性CD44高表达T细胞；对CD8阳性肿瘤浸润T细胞开展单细胞靶向RNA测序联合T细胞受体测序（single-cell targeted RNA sequencing combined with TCR sequencing, scRNA/TCRseq）；对肿瘤引流淋巴结中的CD8阳性CD44高表达T细胞进行批量T细胞受体测序；最终鉴定出肿瘤与肿瘤引流淋巴结之间共有的T细胞克隆。