IL-7 receptor alpha defines heterogeneity and signature of human effector memory CD8+ T cells in high dimensional analysis
Introduction
Distinct subsets of CD4+ and CD8+ T cells are identified based on the expression of molecules including co-stimulatory molecules and receptors for cytokines and chemokines. CD4+ T helper (Th) 1, Th2 and Th17 differentially express CXCR3, CCR4 and CCR6 [1] while IL-1 receptor (R) I can define human Th17 cells [2]. In CD8+ T cells, the high affinity receptor alpha chain for IL-7 or IL-7Rα is differentially expressed by human effector memory (EM) CD8+ T cells, allowing the identification of IL-7Rαlow and high EM CD8+ T cells with distinct cellular characteristics such as the expression of cytotoxic molecules, cytokines, co-stimulatory molecules, and DNA methylation [3], [4], [5], [6].
The introduction of high-dimensional single cell analysis has revolutionized the understanding of cellular heterogeneity as determined by gene and protein expression. Single-cell RNA-seq (sc-RNA-seq) can be used to identify complex and rare cell populations and dissect the relationship among such cell populations [7]. Mass cytometry or Cytometry by Time-Of-Flight (CyTOF) is a recently developed technology for multiparameter single cell analysis that permits unique high dimensional cell profiling at protein levels [8], [9], [10]. CyTOF utilizes heavy metal ions and mass spectrometry as labels and a readout, respectively, which allows the measuring of up to 40 + molecules in a single tube [11], [12], [13]. The high-dimensional CyTOF and scRNA-seq data can be analyzed to assess the multidimensional relationships of molecules expressed by single cells using computational methods such as dimensionality reduction and clustering algorithms [7], [8], [9]. The dimensionality reduction method of principal component analysis (PCA) was applied in analyzing human peripheral CD8+ T cells, showing diverse characteristics of total and viral specific CD8+ T cells [14], [15], [16], [17]. Also, the relationships in multidimensional data can be visualized using the nonlinear dimensionality-reduction tool t-distributed stochastic neighbor embedding (t-SNE) that is used in combination with clustering analysis to robustly identify cellular subsets with distinct traits [18], [19]. Cellular development trajectories on multidimensional CyTOF data can be constructed using the method Wanderlust [20]. Also, the strength of the relationship among molecules expressed by cells can be quantified at the protein level via the application of the algorithm conditional-Density Resampled Estimate of Mutual Information (DREMI) on CyTOF data [21]. The function underlying the relationship can be visualized by conditional-Density Rescaled Visualization (DREVI) [21].
Human EM CD8+ T cells are divided into a few subsets based on the expression of a single or two molecules like CD27, CD28, CD45RA and IL-7Rα [3], [22], [23]. Previously, we showed the presence of human IL-7Rαhigh and low EM CD8+ T cell subsets with distinct cellular characteristics [3], [4], [5], [6]. However, it is unknown whether IL-7Rα can serve as a key molecule in defining a comprehensive landscape of heterogeneity in human EM CD8+ T cells as determined by high-dimensional single cell analysis. Here we addressed this question by analyzing CyTOF and scRNA-seq data of human EM CD8+ T cells using computational algorithms. The results of our study show heterogeneity in human EM CD8+ T cells as defined by distinct but organized expression patterns of multiple molecules, the unique position of IL-7Rα in defining such heterogeneity, and possible biological significance of differential expression of these molecules in modulating downstream events.
Section snippets
Human subjects
Peripheral blood was obtained after informed consent from healthy adult donors as done previously [2], [24], [25]. This work was approved by the institutional review committee of Yale University.
CyTOF analysis
Peripheral blood mononuclear cells (PBMCs) were purified from blood using FicollPAQUE gradients. CyTOF analysis was done as previously done with some modifications [26]. All mass cytometry reagents were purchased from Fluidigm, Inc (South San Francisco, CA) unless otherwise noted. PBMCs (2 × 106 cells)
High-dimensional CyTOF analysis shows heterogeneity in human EM CD8+ T cells expressing different levels of IL-7 receptor alpha chain
We determined the expression of a set of 21 surface molecules, including IL-7Rα, in human EM CD8+ T cells using CyTOF to investigate the implication of IL-7Rα in defining cellular heterogeneity in the setting of high-dimensional profiling. These molecules included the ones encoded by differentially expressed genes (DEGs) in IL-7Rαlow and high EM CD8+ T cells [31] as well as those related to cell activation, co-stimulation, inhibition and migration. To characterize cell subsets in multiple
Discussion
We investigated the role of IL-7Rα in defining heterogeneity of human EM CD8+ T cells and its expressional relationship with molecules implicated in such heterogeneity using high-dimensional CyTOF and scRNA-seq analyses. The results of our study showed that IL-7Rα has diverse but organized expressional relationship with molecules related to gene regulation, effector function, survival and migration in human EM CD8+ T cells, rendering a landscape of heterogeneous EM CD8+ T cell subsets. Such
Funding sources
This work was supported in part by grants from the National Institutes of Health (2R56AG0280691, 1R01AG056728, and R21AI126604 to IK; K24AG042489, P30AG021342 (Yale Claude D. Pepper Older Americans Independence Center) to ACS; and R01AG055362 to IK and ACS).
CRediT authorship contribution statement
Min Sun Shin: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Visualization. Dongjoo Kim: Investigation, Formal analysis, Writing - original draft. Kristina Yim: Methodology, Formal analysis. Hong-Jai Park: Methodology, Writing - review & editing. Sungyong You: Methodology, Writing - review & editing. Xuemei Dong: Resources, Writing - review & editing. Fotios Koumpouras: Resources, Writing - review & editing. Albert C. Shaw: Resources, Writing - review
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank Drs. Steven Kleinstein and Bram Gerritsen for their constructive discussions and critical review of the manuscript. We also thank Dr. Ala Nassar and Ms. Shelly Ren of the Yale CyTOF Core.
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