Single-cell technologies to study the immune system

Abstract

The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system.

Keywords: CD4+ T helper cells; immune cells; single-cell RNA-sequencing; single-cell technology.

Figure 1

The complexity of the blood cell populations has grown in parallel with the development of always more sophisticated technology. From the discovery of red blood cells in 1658 by the Dutch naturalist, Jan Swammerdam, almost 200 years passed until the identification of leucocytes (1843) by two independent physicians from England and France establishing the beginning of haematology as a new field in medicine. The molecular characterization of the leucocytes required the advent of flow cytometry (1960) and monoclonal antibodies (1975). The latter were a crucial tool for the discrimination of CD4⁺ and CD8⁺ T helper cells. In the next decades the scenario of CD4⁺ T helper cells became more and more complicated with the discovery of distinct subclasses. In 1986 Mosman and Coffman revealed the existence of two functional subsets, termed T helper 1 (Th1) and T helper 2 (Th2). In 1995 Dr Sakaguchi60, 61 discovered another specific subpopulation of T cells, named regulatory T (Treg) cells, that were specialized for immunosuppression. More recently other subsets have been isolated named Th17 (2005),62, 63 Th9 (2008)64, 65 and Th22 (2009).40 Finally, single‐cell RNA‐sequencing has revealed the existence of a subpopulation of steroid‐producing cells within the Th2 compartment.23

Figure 2

Identification and characterisation of novel immune cell types and cell states (1) Identification of novel immune cell populations or distinct cell states can be performed using hierarchical clustering (1A) or principle component analysis (PCA) for example (1B). (2) Analysis of differential splicing: specific splice variants may associate with a subpopulation of immune cells or cell state because of their differential function (2A). Example of different approaches to characterise novel cell states. Find markers of cell types by analysing differential expression between different groups of cells (2B), identification of genes that show particular pattern during differentiation such as during developmental maturation of immune cells or in response to immunogenic stimuli: genes that either increase, decrease or are transiently expressed (2C).

Figure 3

The static and discrete view of the CD4+ T helper cell population composition might be completely revised thanks to single‐cell technology. In the new scenario many more intermediate subtypes as well as new subpopulations can be introduced by the whole transcriptomic profiles of single immune cells. In this scheme, CD4+ T helper cells are used as an example that can be applied to many different cell types.