Induction of neoantigen-reactive T cells from healthy donors
Induction of neoantigen-reactive T cells from healthy donors
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DOI：
10.1038/s41596-019-0170-6
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发表时间：
2019-06-01
期刊：
影响因子：
14.8
通讯作者：
Olweus, Johanna
中科院分区：
文献类型：
--
作者：
Ali, Muhammad;Foldvari, Zsofia;Olweus, Johanna
免疫原性新抗原及其同源T细胞的鉴定代表了基于疫苗接种或T细胞受体（TCR）工程化T细胞输注的个性化癌症免疫疗法开发中最关键和限速的步骤。深度测序技术和计算机预测算法的最新进展允许快速鉴定候选新表位。然而，由于患者材料的有限可用性和新表位特异性T细胞的低频率，推定的新表位的大规模验证和反应性T细胞的分离具有挑战性。在这里，我们描述了一个标准化的协议，从健康供体T细胞库的新表位反应性T细胞的诱导，不受潜在的免疫抑制环境的荷瘤宿主。用编码候选新表位的mRNA转染的单核细胞衍生的树突状细胞（DC）用于引发自体初始CD 8（+）T细胞。使用肽-MHC（pMHC）多聚体检测识别内源性加工和呈递的表位的抗原特异性T细胞。在包括克隆扩增和功能表征的任选步骤之后，分选单个多聚体阳性T细胞以鉴定TCR序列。鉴定新表位特异性T细胞所需的时间为15天，克隆扩增和下游功能表征需要额外的2-4周。鉴定的新表位和相应的TCR提供了用于疫苗接种和基于TCR的癌症免疫疗法的候选物，并且通过该技术生成的数据集应该可用于改进预测免疫原性新抗原的算法。
The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitopereactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8(+) T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.