Tumor-infiltrating lymphocyte(TIL)therapy was recently approved for melanoma patients;however,the dynamic changes in T cell subpopulations during TIL production remain poorly understood.Here,we analyzed epithelial ova...Tumor-infiltrating lymphocyte(TIL)therapy was recently approved for melanoma patients;however,the dynamic changes in T cell subpopulations during TIL production remain poorly understood.Here,we analyzed epithelial ovarian cancer samples at various stages of ex vivo TIL culture using paired single-cell RNA and TCR sequencing.We also assessed the expansion potential and tumor reactivity of the identified TIL subpopulations.Single-cell transcriptomic analysis revealed that CD8^(+) TILs exhibited reduced cellular diversity following ex vivo expansion,selectively expanding stem-like TCF7^(+) precursors of exhausted T cells(Tpex)and effector-like tissue-resident memory(Trm)cells.TCR clonotype analysis showed that Tpex cells accumulated through self-renewal,while Trm cells primarily originated from TCF7^(+)GZMK+early effector memory cells in tumors.Additionally,TCR tracing identified preferential activation and reprogramming of CD4^(+)T follicular helper(Tfh)-like cells,especially TCF7+ones.All three TCF7+subpopulations showed robust expansion potential and tumor reactivity in vitro.Notably,CCR7^(+)CD200^(+)T cells,enriched for TCF-1+CD8^(+)Tpex and CD4^(+)Tfh-like cells in the tumor microenvironment,exhibited self-renewal during in vitro expansion and demonstrated tumor reactivity both in vivo and in vitro.These findings highlight the selective expansion of tumor-reactive TCF7^(+)T cells during TIL culture and suggest that CCR7 and CD200 serve as important surface markers for generating stem-like,tumor-reactive cells,potentially improving TIL therapy in cancers.展开更多
基金supported by the National Key Research and Development Program of China(2021YFC2302403,2022YFC2704703,2022YFC2702204,2024YFA1306001)the National Natural Science Foundation of China(31991173,31991170,82271701,81730039,82071653,82102853,82372888)+2 种基金the Shanghai Pudong New Area Health Commission Project(PW2022D-05)Peking University Clinical Scientist Training Program of the Fundamental Research Funds for the Central Universities(BMU2024PYJH008)supported by a New Cornerstone Investigator award.
文摘Tumor-infiltrating lymphocyte(TIL)therapy was recently approved for melanoma patients;however,the dynamic changes in T cell subpopulations during TIL production remain poorly understood.Here,we analyzed epithelial ovarian cancer samples at various stages of ex vivo TIL culture using paired single-cell RNA and TCR sequencing.We also assessed the expansion potential and tumor reactivity of the identified TIL subpopulations.Single-cell transcriptomic analysis revealed that CD8^(+) TILs exhibited reduced cellular diversity following ex vivo expansion,selectively expanding stem-like TCF7^(+) precursors of exhausted T cells(Tpex)and effector-like tissue-resident memory(Trm)cells.TCR clonotype analysis showed that Tpex cells accumulated through self-renewal,while Trm cells primarily originated from TCF7^(+)GZMK+early effector memory cells in tumors.Additionally,TCR tracing identified preferential activation and reprogramming of CD4^(+)T follicular helper(Tfh)-like cells,especially TCF7+ones.All three TCF7+subpopulations showed robust expansion potential and tumor reactivity in vitro.Notably,CCR7^(+)CD200^(+)T cells,enriched for TCF-1+CD8^(+)Tpex and CD4^(+)Tfh-like cells in the tumor microenvironment,exhibited self-renewal during in vitro expansion and demonstrated tumor reactivity both in vivo and in vitro.These findings highlight the selective expansion of tumor-reactive TCF7^(+)T cells during TIL culture and suggest that CCR7 and CD200 serve as important surface markers for generating stem-like,tumor-reactive cells,potentially improving TIL therapy in cancers.
文摘寨卡病毒(Zika virus,ZIKV)是一种蚊媒黄病毒,也可通过性传播,同时还能感染睾丸组织并导致损伤.探究寨卡病毒在睾丸内持续复制过程中病毒基因的变化规律,发现潜在的睾丸适应性突变位点,可为寨卡病毒变异的风险评估提供重要参考.为此,本研究以寨卡病毒株FSS13025为研究对象,通过将其在A129小鼠睾丸中连续传代10轮后获得适应性传代毒株,将原始株(WT)、第5代(P5)和第10代(P10)病毒经腹腔接种A129小鼠后发现,睾丸中P5和P10的RNA载量较WT分别提高4.6和3.8倍;进一步通过深度测序分析P5和P10的基因组变异情况,结果显示二者的包膜蛋白(envelope,E)和非结构蛋白1(nonstructural protein 1,NS1)分别出现H401Y和K146E突变.在此基础上,以寨卡病毒FSS13025株为基因骨架,利用反向遗传学技术构建拯救携带上述突变的重组病毒H401Y+K146E,并评价其在细胞和小鼠体内的复制能力,结果显示病毒感染睾丸细胞15P-1和TM3后第48 h,细胞培养上清中的重组病毒RNA含量分别是WT的5.2和2.1倍;经腹腔途径感染A129小鼠后的第3和6天,睾丸中重组病毒RNA载量较WT分别提高97.5和58.4倍.上述结果表明,H401Y+K146E联合突变显著增强了寨卡病毒在睾丸细胞中的复制能力,同时明显提升了其对睾丸的嗜性和侵袭力,上述位点可作为寨卡病毒变异风险预警的监测靶点.
