Cellular heterogeneity of circulating CD4(+)CD8(+) double-positive T cells characterized by single-cell RNA sequencing

Circulating CD4(+)CD8(+) double-positive (DP) T cells are associated with a variety of disease states. However, unlike conventional T cells, the composition of this population is poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to analyze the composition and characteristics of the DP T cell population circulating in the peripheral blood of cynomolgus monkeys. We found that circulating DP T cells not only contain a large number of naïve cells, but also comprise a heterogeneous population (CD4 CTL-, Eomes(+) Tr1-, Th2-, Th17-, Tfh-, Treg-, CD8 CTL-, and innate-like cells) with multiple potential functions. Flow cytometry analysis revealed that a substantial number of the naïve DP T cells expressed CD8αβ, as well as CD8αα, along with high expression of CD31. Moreover, the CD4(hi)CD8(lo) and CD4(hi)CD8(hi) populations, which express high levels of the CD4 coreceptor, comprised subsets characterized by helper and regulatory functions, some of which also exhibited cytotoxic functions. By contrast, the CD4(lo)CD8(hi) population with high CD8 coreceptor expression comprised a subset characterized by CD8 CTL- and innate-like properties. Taken together, the data show that scRNA-seq analysis identified a more diverse subset of the circulating DP cells than is currently known, despite this population being very small.