Single-Cell RNA-Sequencing Identifies Activation of TP53 and STAT1 Pathways in Human T Lymphocyte Subpopulations in Response to Ex Vivo Radiation Exposure.

Honglu Wu,Winston Wu,Mayra Nelman-Gonzalez,Sujash Chatterjee,Maria Moreno-Villanueva,Alan Feiveson,Stephanie Krieger,Ryan Clanton,Preethi Gunaratne,Brandon Mistretta,Brian Crucian,Yinghong Pan,Ye Zhang

doi:10.3390/ijms20092316

Abstract

Detrimental health consequences from exposure to space radiation are a major concern for long-duration human exploration missions to the Moon or Mars. Cellular responses to radiation are expected to be heterogeneous for space radiation exposure, where only high-energy protons and other particles traverse a fraction of the cells. Therefore, assessing DNA damage and DNA damage response in individual cells is crucial in understanding the mechanisms by which cells respond to different particle types and energies in space. In this project, we identified a cell-specific signature for radiation response by using single-cell transcriptomics of human lymphocyte subpopulations. We investigated gene expression in individual human T lymphocytes 3 h after ex vivo exposure to 2-Gy gamma rays while using the single-cell sequencing technique (10X Genomics). In the process, RNA was isolated from ~700 irradiated and ~700 non-irradiated control cells, and then sequenced with ~50 k reads/cell. RNA in each of the cells was distinctively barcoded prior to extraction to allow for quantification for individual cells. Principal component and clustering analysis of the unique molecular identifier (UMI) counts classified the cells into three groups or sub-types, which correspond to CD4+, naïve, and CD8+/NK cells. Gene expression changes after radiation exposure were evaluated using negative binomial regression. On average, BBC3, PCNA, and other TP53 related genes that are known to respond to radiation in human T cells showed increased activation. While most of the TP53 responsive genes were upregulated in all groups of cells, the expressions of IRF1, STAT1, and BATF were only upregulated in the CD4+ and naïve groups, but were unchanged in the CD8+/NK group, which suggests that the interferon-gamma pathway does not respond to radiation in CD8+/NK cells. Thus, single-cell RNA sequencing technique was useful for simultaneously identifying the expression of a set of genes in individual cells and T lymphocyte subpopulation after gamma radiation exposure. The degree of dependence of UMI counts between pairs of upregulated genes was also evaluated to construct a similarity matrix for cluster analysis. The cluster analysis identified a group of TP53-responsive genes and a group of genes that are involved in the interferon gamma pathway, which demonstrate the potential of this method for identifying previously unknown groups of genes with similar expression patterns.

Highlights

In space, astronauts are constantly exposed to cosmic radiation that consists of energetic charged particles [1]
Radiation-induced gene expression has been assessed in populations of cultured cells or in animal tissues, while chromosome aberrations and DNA strand breaks can be analyzed in individual cells [7,8,9,10]
We intended to only isolate T cells, some natural killer (NK) cells were included in the cell population studied

Summary

Introduction

Astronauts are constantly exposed to cosmic radiation that consists of energetic charged particles [1]. The cellular effects of space radiation exposure, at the molecular level, have far been assessed mostly in pools of cells that have been irradiated with a single particle type [2]. In the space environment, only a small fraction of the cells would be traversed by particles at a given time. In such an environment, the cells may experience severe radiation damage, possibly leading to chromosomal aberrations or cell death [3,4]. It is necessary to investigate the cellular responses to space radiation exposure in individual cells in order to accurately assess the associated health risks. Single cells can be captured in an oil droplet containing one hydrogel bead (gel bead) that is labeled with unique barcoded oligonucleotides that tag individual cells [13]. scRNA-seq has been applied in multiple research fields, such as in cancer biology for exploring tumor cell heterogeneity, in immunology for identifying new T cell receptors, or in neuroscience for investigating neural cell diversity [14,15,16]

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