T cells are important in tumour immunity but a better understanding is needed of the differentiation of antigen-specific T cells in human cancer^1,2. Here we studied CD8 T cells in patients with human papillomavirus (HPV)-positive head and neck cancer and identified several epitopes derived from HPV E2, E5 and E6 proteins that allowed us to analyse virus-specific CD8 T cells using major histocompatibility complex (MHC) class I tetramers. HPV-specific CD8 T cells expressed PD-1 and were detectable in the tumour at levels that ranged from 0.1% to 10% of tumour-infiltrating CD8 T lymphocytes (TILs) for a given epitope. Single-cell RNA-sequencing analyses of tetramer-sorted HPV-specific PD-1⁺ CD8 TILs revealed three transcriptionally distinct subsets. One subset expressed TCF7 and other genes associated with PD-1⁺ stem-like CD8 T cells that are critical for maintaining T cell responses in conditions of antigen persistence. The second subset expressed more effector molecules, representing a transitory cell population, and the third subset was characterized by a terminally differentiated gene signature. T cell receptor clonotypes were shared between the three subsets and pseudotime analysis suggested a hypothetical differentiation trajectory from stem-like to transitory to terminally differentiated cells. More notably, HPV-specific PD-1⁺TCF-1⁺ stem-like TILs proliferated and differentiated into more effector-like cells after in vitro stimulation with the cognate HPV peptide, whereas the more terminally differentiated cells did not proliferate. The presence of functional HPV-specific PD-1⁺TCF-1⁺CD45RO⁺ stem-like CD8 T cells with proliferative capacity shows that the cellular machinery to respond to PD-1 blockade exists in HPV-positive head and neck cancer, supporting the further investigation of PD-1 targeted therapies in this malignancy. Furthermore, HPV therapeutic vaccination efforts have focused on E6 and E7 proteins; our results suggest that E2 and E5 should also be considered for inclusion as vaccine antigens to elicit tumour-reactive CD8 T cell responses of maximal breadth.

T 细胞在肿瘤免疫中很重要，但需要更好地了解人类癌症中抗原特异性 T 细胞的分化^1,2。在这里，我们研究了人乳头瘤病毒 (HPV) 阳性头颈癌患者的 CD8 T 细胞，并鉴定了几个源自 HPV E2、E5 和 E6 蛋白的表位，使我们能够使用主要组织相容性复合体 (MHC) 分析病毒特异性 CD8 T 细胞) I 类四聚体。HPV 特异性 CD8 T 细胞表达 PD-1，并且在肿瘤中可检测到的水平范围为给定表位的肿瘤浸润 CD8 T 淋巴细胞 (TIL) 的 0.1% 至 10%。^{四聚体分类的 HPV 特异性 PD-1 +} CD8 TIL的单细胞 RNA 测序分析揭示了三个转录不同的亚群。一个子集表达了TCF7^{以及与 PD-1 +}干细胞样 CD8 T 细胞相关的其他基因，这些细胞对于在抗原持久性条件下维持 T 细胞反应至关重要。第二个子集表达更多的效应分子，代表一个短暂的细胞群，第三个子集的特征是终末分化的基因特征。T 细胞受体克隆型在三个亚群之间共享，假时间分析表明从干细胞样细胞到过渡细胞再到终末分化细胞的假设分化轨迹。更值得注意的是，HPV 特异性 PD-1 ⁺ TCF-1 ⁺在体外用同源 HPV 肽刺激后，干细胞样 TIL 增殖并分化成更多的效应样细胞，而更终末分化的细胞没有增殖。功能性 HPV 特异性 PD-1 ⁺ TCF-1 ⁺ CD45RO ⁺具有增殖能力的干细胞样 CD8 T 细胞的存在表明，响应 PD-1 阻断的细胞机制存在于 HPV 阳性头颈癌中，支持PD-1 靶向治疗在这种恶性肿瘤中的进一步研究。此外，HPV 治疗性疫苗接种工作的重点是 E6 和 E7 蛋白；我们的结果表明，E2 和 E5 也应考虑作为疫苗抗原包含在内，以引发最大宽度的肿瘤反应性 CD8 T 细胞反应。