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^hiCD8^lo and CD4^hiCD8^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^loCD8^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.