Multi-omic analyses in immune cell development with lessons learned from T cell development

Abstract

Traditionally, flow cytometry has been the preferred method to characterize immune cells at the single-cell level. Flow cytometry is used in immunology mostly to measure the expression of identifying markers on the cell surface, but-with good antibodies-can also be used to assess the expression of intracellular proteins. The advent of single-cell RNA-sequencing has paved the road to study immune development at an unprecedented resolution. Single-cell RNA-sequencing studies have not only allowed us to efficiently chart the make-up of heterogeneous tissues, including their most rare cell populations, it also increasingly contributes to our understanding how different omics modalities interplay at a single cell resolution. Particularly for investigating the immune system, this means that these single-cell techniques can be integrated to combine and correlate RNA and protein data at the single-cell level. While RNA data usually reveals a large heterogeneity of a given population identified solely by a combination of surface protein markers, the integration of different omics modalities at a single cell resolution is expected to greatly contribute to our understanding of the immune system.

Keywords: T cells; multi‐omics; scRNA‐seq; spectral flow cytometry; thymus.

FIGURE 1

Overview of T cell development in the human thymus. Cross section of an adult thymic lobule representing the migration route of T-cell precursors during development. Immigrant precursors move to the thymus through blood vessels and enter near the corticomedullary junction; the TSPs subsequently migrate, and differentiate to DN, DP and finally to SP stages through the discrete microenvironments of the thymus. A directional reversal of migration back across the cortex towards the medulla occurs for the later stages of thymocyte development. The cell percentages represent the proportions of cell types compared to the total cell composition in the human thymus. DN: Double negative; DP: Double positive; ISP: Immature single positive; SP: Single positive; TSP: Thymic seeding progenitor.

FIGURE 2

An example of a multi-omics approach to understand human T cell development (Cordes et al., 2022). Discovery: Purification of thymocyte subsets isolated from 6 healthy donors to generate a multi-omic (gene expression and γδ/αβ TCR) scRNAseq dataset resulting in the identification of different thymus seeding progenitor populations and detection of multiple lineage differentiation trajectories (including alternative lineages). Validation: by functional assays, lineage tracing and multi-color flow cytometry.

FIGURE 3

Schematic overview of the main steps and considerations used to perform studies on rare cells.