Zoutman Willem H, Nell Rogier J, van der Velden Pieter A
Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands.
Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
Methods Mol Biol. 2019;1884:1-14. doi: 10.1007/978-1-4939-8885-3_1.
T cells fulfill a central role in cell-mediated immunity and can be found in the circulation and lymphoid organs upon maturation. For clinical applications, it can be important to quantify (infiltrated) T cells accurately in a variety of body fluids and tissues of benign, inflammatory, or malignant origin. For decades, flow cytometry and immunohistochemistry have been the accustomed methods to quantify T cells. Although these methods are widely used, they depend on the accessibility of T-cell epitopes and therefore require fresh, frozen, or fixated material of a certain quality. Whenever samples are low in quantity or quality, an accurate quantification can be impeded. By shifting the focus from epitopes to DNA, quantification of T cells remains achievable.Mature T cells differ genetically from other cell types as a result of T-cell receptor (TCR) gene rearrangements. This genetic dissimilarity can be exploited to quantify the T-cell fraction in DNA specimens. Conventionally, multiplex PCR and droplet digital PCR (ddPCR), combined with deep-sequencing techniques, can be applied to determine T-cell content. However, these approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. Considering this, a simple T-cell quantification, unwantedly, turns into a complex, expensive, and time-consuming procedure. We have developed two generic single duplex ddPCR assays as alternative methods to quantify T cells in a relatively simple, cheap, and fast manner by targeting sequences located between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus). These specific TCR loci become deleted systematically early during lymphoid differentiation and therefore will serve as biomarkers for the quantification of mature T cells. Here, we describe a simple and sensitive ddPCR-based method to quantify T cells relatively fast, accurately and independently of the cellular context.
T细胞在细胞介导的免疫中发挥核心作用，成熟后可在循环系统和淋巴器官中找到。对于临床应用而言，准确量化各种良性、炎症性或恶性来源的体液和组织中的（浸润）T细胞可能非常重要。几十年来，流式细胞术和免疫组织化学一直是量化T细胞的常用方法。尽管这些方法被广泛使用，但它们依赖于T细胞表位的可及性，因此需要一定质量的新鲜、冷冻或固定材料。每当样本数量或质量不足时，准确的量化就会受到阻碍。通过将重点从表位转移到DNA，仍然可以实现T细胞的量化。由于T细胞受体（TCR）基因重排，成熟T细胞在基因上与其他细胞类型不同。这种基因差异可用于量化DNA样本中的T细胞比例。传统上，多重PCR和液滴数字PCR（ddPCR）结合深度测序技术可用于确定T细胞含量。然而，这些方法通常针对整个TCR库，从而提供有关TCR使用的额外信息。考虑到这一点，一个简单的T细胞量化意外地变成了一个复杂、昂贵且耗时的过程。我们开发了两种通用的单重ddPCR检测方法，作为以相对简单、廉价和快速的方式量化T细胞的替代方法，通过靶向位于Dδ2和Dδ3基因（TRD基因座）以及Dβ1和Jβ1.1基因（TRB基因座）之间的序列。这些特定的TCR基因座在淋巴分化早期会系统性地缺失，因此将作为成熟T细胞量化的生物标志物。在这里，我们描述了一种基于ddPCR的简单且灵敏的方法，可相对快速、准确且独立于细胞背景地量化T细胞。
