Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
Charité-Universitätsmedizin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.
Methods Mol Biol. 2022;2521:95-128. doi: 10.1007/978-1-0716-2441-8_6.
The protocol describes the procedure of antigen-specific T cell generation and TCR identification for the use in adoptive T cell therapy. We describe two paths of generating antigen-specific T cells, first, T cell stimulation with autologous dendritic cells pulsed with antigen peptide, second, in vivo T cell stimulation with peptide or DNA by gene gun application in a suitable mouse model followed by in vitro enrichment of peptide-reactive T cells. Peptide-stimulated T cells are sorted by fluorescence-activated cell sorting for CD107α or IFNγ expression and subsequently isolated RNA is used in a 5' rapid amplification of cDNA ends (RACE )-PCR specific for TCR for TCR chain identification. After retroviral cloning, it is re-expressed on human T cells to test its applicability in adoptive T cell therapy.
该方案描述了用于过继性 T 细胞治疗的抗原特异性 T 细胞生成和 TCR 鉴定的程序。我们描述了两种生成抗原特异性 T 细胞的途径，首先，用抗原肽脉冲处理的自体树突细胞刺激 T 细胞，其次，通过基因枪应用于合适的小鼠模型进行体内 T 细胞刺激，然后体外富集肽反应性 T 细胞。用荧光激活细胞分选术对肽刺激的 T 细胞进行 CD107α 或 IFNγ 表达的分选，随后分离 RNA 并用于 5'快速扩增 cDNA 末端 (RACE)-PCR 特异性 TCR 以鉴定 TCR 链。在逆转录病毒克隆后，它在人 T 细胞上重新表达，以测试其在过继性 T 细胞治疗中的适用性。
