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

Sung Min Choi,Jae Il Lee,Hi Jung Park,Eun A Choi,Kyeong Cheon Jung

doi:10.1038/s41598-021-03013-4

Abstract

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 CD4hiCD8lo and CD4hiCD8hi 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 CD4loCD8hi 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.

Highlights

The unbiased and high-throughput nature of modern single-cell RNA sequencing approaches has proven invaluable for describing heterogeneous cell populations[1,2]
A uniform manifold approximation and projection (UMAP) of sorted DP T cells showed that CD3E, CD4 FB, and CD8 FB were highly expressed by all clusters, whereas NCAM1 (CD56) and TRAV24 (TCRVα24) were expressed at either very low levels or were absent (Fig. 1b)
Compared with single-positive T cells, DP T cells exhibit pleiotropic features, including helper, cytotoxic, regulatory, and innate-like roles, depending on their expression of CD4 or CD8 coreceptors. These DP T cells can be developed by a variety of pathways, such as thymocyte-like DP T cells have observed in certain pathologic conditions[37,38]

Summary

Introduction

The unbiased and high-throughput nature of modern single-cell RNA sequencing (scRNA-seq) approaches has proven invaluable for describing heterogeneous cell populations[1,2]. Cellular indexing of transcriptomes and epitopes by sequencing, a method in which oligonucleotide-labeled antibodies are used to integrate cellular protein and transcriptome measurements into an efficient single-cell readout, was described r ecently[3] This process provides a detailed understanding of limited T cell frequencies at the single-cell level, while being able to reveal the broad heterogeneity of a specific T cell subset. In a previous study[4], we found that some DP T cells express significant levels of promyelocytic leukemia zinc finger (PLZF)[15] and eomesodermin (Eomes)[16], both markers of innate lymphocytes, when compared with CD4 SP and CD8 SP T cells. We used the scRNA-seq platform to analyze the characteristics of circulating DP T cells and to compare their transcriptomic profiles with their cellular phenotypes and cytokine secretion patterns

Methods

Results

Conclusion