Department of Hematology, Leiden University Medical Center, The Netherlands.
Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory for Blood Cell Research, Amsterdam, The Netherlands.
J Infect Dis. 2022 Sep 13;226(5):833-842. doi: 10.1093/infdis/jiaa512.
Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far.
HLA-A01:01-restricted EBV-LMP2-specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-γ) and cytotoxicity when stimulated with EBV-LMP2-expressing cell lines. Functionality of primary T cells transduced with HLA-A01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells (∆TCR) using CRISPR-Cas9 technology.
EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2-expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5-2 fold when the endogenous TCRs of CD8+ T cells was knocked out. CD8+/∆TCR T cells modified to express EBV-LMP2-specific TCRs showed IFN-γ secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines.
We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.
表达抗原特异性 T 细胞受体（TCR）的基因工程 T 细胞的过继转移是一种很有吸引力的治疗方法，适用于表达 EBV 抗原（LMP1/2）的潜伏 II/III 型 EBV 相关恶性肿瘤。HLA-A*01:01 阳性的患者可以从这些产品中受益，因为到目前为止还没有发现能够识别这种常见 HLA 等位基因中任何 EBV 衍生肽的 T 细胞。
使用肽主要组织相容性复合物（pMHC）四聚体分离 HLA-A01:01 限制性 EBV-LMP2 特异性 T 细胞。通过 IFN-γ的产生和与表达 EBV-LMP2 的细胞系刺激时的细胞毒性来评估功能。使用 CRISPR-Cas9 技术敲除原代 T 细胞的内源性 TCR（∆TCR），优化转导 HLA-A01:01 限制性 EBV-LMP2 特异性 TCR 的原代 T 细胞的功能。
成功分离出 EBV-LMP2 特异性 T 细胞，并对其 TCR 进行了表征。TCR 基因转移到原代 T 细胞中导致特异性 pMHC 四聚体结合，并对表达 EBV-LMP2 的细胞系产生反应。当 CD8+T 细胞的内源性 TCR 被敲除时，pMHC-四聚体结合的平均荧光强度增加了 1.5-2 倍。表达 EBV-LMP2 特异性 TCR 的 CD8+/∆TCR T 细胞表现出 IFN-γ分泌和对表达 EBV-LMP2 的恶性细胞系的细胞毒性。
我们分离出了第一个功能性 HLA-A*01:01 限制性 EBV-LMP2 特异性 T 细胞群和 TCR，它们有可能在未来的 TCR 基因治疗中用于治疗 EBV 相关的潜伏 II/III 型恶性肿瘤。
