Abstract：
Adoptive transfer of T-cell receptor (TCR)-engineered T cells has been successful in mediating favorable clinical outcomes. TCR-engineered T cells can be applied for targeting cancers whose associated antigens are intracellular and presented through major histocompatibility complexes (MHC). The mispairing of the exogenous TCR chains with the endogenous TCR chains leads to functionally impaired TCR-engineered T cells. The CRISPR/Cas9 genome-editing system can be utilized for the knockout of the endogenous TCR in T cells before introducing the exogenous TCR chains. In this study, we used the lentiviral delivery of CRISPR/Cas9 for disrupting the expression of the endogenous TCR in the Jurkat cell line. Next, an exogenous TCR targeting human leukocyte antigen (HLA)-A*0201-restricted New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide was transduced into the TCR-knockout (KO) Jurkat cells. Further, we assessed lentiviral transduction efficacy using tetramer assay and evaluated the functionality of the NY-ESO-1-specific TCR-engineered T cells by quantifying the cell surface expression of CD69 upon co-cultivation with peptide-pulsed T2 cells. We successfully knocked out the endogenous TCR in ∼40% of the Jurkat cells. TCR-KO cells were selected and subjected to express NY-ESO-1-specific TCRs using lentiviral vectors. Flow cytometry analysis confirmed that up to 55% of the cells expressed the transgenic TCR on their surface. The functionality assay demonstrated that >90% of the engineered cells expressed CD69 when co-cultured with peptide-pulsed T2 cells. Conclusively, we developed a pipeline to engineer Jurkat cells using the state-of-the-art technique CRISPR/Cas9 and generated TCR-engineered cells that can become activated by a tumor-specific antigen.
摘要：
T细胞受体(TCR)工程化的T细胞的过继转移已经成功地介导了有利的临床结果。TCR工程化的T细胞可用于靶向其相关抗原在细胞内并通过主要组织相容性复合物(MHC)呈递的癌症。外源性TCR链与内源性TCR链的错配导致功能性受损的TCR工程化T细胞。CRISPR/Cas9基因组编辑系统可用于在引入外源TCR链之前敲除T细胞中的内源TCR。在这项研究中,我们使用CRISPR/Cas9的慢病毒递送来破坏Jurkat细胞系中内源性TCR的表达。接下来,将外源性TCR靶向人类白细胞抗原(HLA)-A*0201限制性纽约食管鳞状细胞癌1(NY-ESO-1)肽转导至TCR敲除(KO)Jurkat细胞.Further,我们使用四聚体测定法评估了慢病毒转导功效,并通过定量与肽脉冲T2细胞共培养后CD69的细胞表面表达,评估了NY-ESO-1特异性TCR工程化T细胞的功能.我们成功敲除了40%的Jurkat细胞中的内源性TCR。选择TCR-KO细胞并使用慢病毒载体表达NY-ESO-1特异性TCR。流式细胞术分析证实高达55%的细胞在其表面上表达转基因TCR。功能性测定表明,当与肽脉冲的T2细胞共培养时,>90%的工程化细胞表达CD69。最后，我们使用最先进的技术CRISPR/Cas9开发了一条工程Jurkat细胞的管道,并产生了可以被肿瘤特异性抗原激活的TCR工程细胞.
