Rollins Meagan R, Spartz Ellen J, Stromnes Ingunn M
Department of Microbiology and Immunology, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota.
Center for Genome Engineering, University of Minnesota Medical School, Minneapolis, Minnesota.
Curr Protoc Immunol. 2020 Jun;129(1):e97. doi: 10.1002/cpim.97.
T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of human autologous dendritic cells from monocytes Basic Protocol 2: In vitro priming and expansion of human antigen-specific T cells Basic Protocol 3: Cloning of antigen-specific T cell receptors based on single-cell VDJ sequencing data Basic Protocol 4: Validation of T cell receptor expression and functionality in vitro Basic Protocol 5: Rapid expansion of T cell receptor-transduced T cells and human T cell clones.
T淋巴细胞能够特异性识别并清除靶细胞。生理性抗原识别由T细胞受体（TCR）介导，TCR是一种αβ异二聚体，由随机重排的V、D和J基因的产物组成。T细胞的高度特异性和功能性可用于癌症治疗：具体而言，过继转移表达肿瘤反应性TCR的T细胞可诱导晚期癌症患者的实体瘤消退。然而，分离和表达肿瘤抗原特异性TCR是一个高度复杂的过程，需要识别免疫原性表位、确保人类细胞具有正确的单倍型、进行费力的T细胞扩增过程，以及进行下游TCR测序和克隆。单细胞测序的最新进展已开始简化这一过程。本方案综合并扩展了相关方法，以生成、分离和改造具有肿瘤反应性TCR的人类T细胞用于过继性细胞治疗。尽管这个过程可能比使用嵌合抗原受体（CAR）进行工程改造的替代策略更艰巨，但使用TCR靶向细胞内蛋白质的能力大大增加了可以安全靶向的抗原类型。© 2020威利期刊有限责任公司。基本方案1：从单核细胞生成人类自体树突状细胞 基本方案2：人类抗原特异性T细胞的体外致敏和扩增 基本方案3：基于单细胞VDJ测序数据克隆抗原特异性T细胞受体 基本方案4：体外验证T细胞受体的表达和功能 基本方案5：T细胞受体转导的T细胞和人类T细胞克隆的快速扩增。
