Department of Melanoma Medical Oncology, Unit 904, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Clin Cancer Res. 2012 Sep 1;18(17):4733-42. doi: 10.1158/1078-0432.CCR-11-3234. Epub 2012 Jul 3.
T-cell receptor (TCR) variable Vα and Vβ gene diversity is a surrogate biomarker for the therapeutic potential of adoptive immunotherapy and cellular immunity. Therefore, creating a straightforward, rapid, sensitive, and reliable method to view the global changes of both TCRVα and Vβ transcripts in heterogeneous populations of T cells is appealing.
We designed a "direct TCR expression assay" (DTEA) using a panel of customized bar-coded probes that simultaneously detects and quantifies 45 Vα and 46 Vβ transcripts in a nonenzymatic digital multiplexed assay from a small number of cells (10(4) cells) or as little as 100 ng of total RNA.
We evaluated DTEA on total RNA samples of tumor-infiltrating lymphocytes and peripheral blood obtained from patients with melanoma after adoptive T-cell therapy. DTEA detected a similar spectrum of the dominant patterns of TCRVβ gene usage as sequencing cloned TCRVβ CDR3 regions. However, DTEA was rapid, achieved a level of sensitivity to identify rare T-cell populations, and simultaneously tracked the full array of Vα and Vβ transcripts.
DTEA can rapidly and sensitively track changes in TCRVα and Vβ gene usages in T-cell pools following immune interventions, such as adoptive T-cell transfer, and may also be used to assess impact of vaccination or reconstitution of T-cell compartment after hematopoietic stem cell transplantation.
T 细胞受体 (TCR) 可变 Vα 和 Vβ 基因多样性是过继免疫治疗和细胞免疫治疗潜力的替代生物标志物。因此，创建一种简单、快速、灵敏和可靠的方法来观察 T 细胞异质性群体中 TCRVα 和 Vβ 转录本的全局变化是吸引人的。
我们设计了一种“直接 TCR 表达测定法”（DTEA），使用一组定制的带条码探针，在非酶促数字多路复用测定中同时检测和定量 45 个 Vα 和 46 个 Vβ 转录本，从少量细胞（10^4 个细胞）或低至 100ng 的总 RNA。
我们在接受过继 T 细胞治疗的黑色素瘤患者的肿瘤浸润淋巴细胞和外周血总 RNA 样本上评估了 DTEA。DTEA 检测到与测序克隆 TCRVβ CDR3 区相似的 TCRVβ 基因使用的优势模式谱。然而，DTEA 快速，达到了识别稀有 T 细胞群体的灵敏度水平，并同时跟踪了全数组的 Vα 和 Vβ 转录本。
DTEA 可以快速、灵敏地跟踪免疫干预（如过继 T 细胞转移）后 T 细胞池 TCRVα 和 Vβ 基因使用的变化，也可用于评估疫苗接种或造血干细胞移植后 T 细胞区的重建对其的影响。
