Heterogeneity of circulating CD4⁺CD8⁺ double-positive T cells characterized by scRNA-seq analysis and trajectory inference

The frequency of CD4⁺CD8⁺ double-positive (DP) T cells is highly associated with a variety of diseases. Recently, we used high-throughput single-cell RNA sequencing to show that circulating DP T cells in cynomolgus monkeys comprise nine heterogeneous populations. To better understand the characteristics of DP T cells, we analyzed 7601 cells from a rhesus monkey and detected 14,459 genes. Rhesus monkey DP T cells comprised heterogeneous populations (naïve, Treg-, Tfh-, CCR9⁺ Th-, Th17-, Th2-, Eomes⁺ Tr1-, CTL-, PLZF⁺ innate- and Eomes⁺ innate-like cells) with multiple potential functions. We also identified two new subsets using aggregated scRNA-seq datasets from the rhesus and the cynomolgus monkey: CCR9⁺ Th-like cells expressing ICAM2 and ITGA1, and PLZF⁺ innate-like cells that display innate-associated gene signatures such as ZBTB16, TYROBP, MAP3K8, and KLRB1. Trajectory inference of cell differentiation status showed that most DP T cells in the rhesus monkey were found in the mid-to-late pseudotime, whereas DP T cells from the cynomolgus monkey were found in early pseudotime. This suggests that DP T cells in rhesus monkeys may exhibit more diverse differentiation states than those in cynomolgus monkeys. Thus, scRNA-seq and trajectory inference identified a more diverse subset of the circulating DP T cells than originally thought.