Usage of Droplet Digital PCR (ddPCR) Assays for T Cell Quantification in Cancer

Abstract

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.