The impact of single-cell RNA sequencing on understanding the functional organization of the immune system

Abstract

Application of single-cell genomics technologies has revolutionized our approach to study the immune system. Unravelling the functional diversity of immune cells and their coordinated response is key to understanding immunity. Single-cell transcriptomics technologies provide high-dimensional assessment of the transcriptional states of immune cells and have been successfully applied to discover new immune cell types, reveal haematopoietic lineages, identify gene modules dictating immune responses and investigate lymphocyte antigen receptor diversity. In this review, we discuss the impact and applications of single-cell RNA sequencing technologies in immunology.

Figure 1.

Heterogeneity of single cells within bulk populations. Cells isolated based on limited number of markers using FACS, by micropipette, optical tweezers or microfluidics, may contain multiple different cell types and cell states (left panel). If these cells are subjected to bulk analysis (squares), their heterogeneity and unique functions are masked by an average of gene expression signals. If studied individually (single cells), then their distinct activation responses to stimuli, such as cytokines, lipopolysaccharide or bacteria, can be uncovered (right panel). A specific stimulus can have variable effect on cells and leads to different outcome readout for the population in total. For instance, the rare cells may be early responders, which differentiate and activate other cells via paracrine signalling, or the cell types may respond differently, by cytokine production (cell type 2/round cells) or differentiation.

Figure 2.

Integrated analysis of the immune system using single-cell RNA-seq. Cells from immune compartments of interest are isolated and analysed by scRNA-seq. During analysis, cells are grouped (clustered) by transcriptome profile. New and known cell clusters can be further investigated. The identity of clusters and authenticity of new cell types can be validated by flow cytometry, RT-qPCR or sequencing of isolated cells. Morphological and functional studies of new cell types or molecules provide additional biological insights. The new knowledge is integrated into subsequent experimental models.