Heterogeneity in immune responses: from populations to single cells

Abstract

The mammalian immune system is tasked with protecting the host against a broad range of threats. Understanding how immune populations leverage cellular diversity to achieve this breadth and flexibility, particularly during dynamic processes such as differentiation and antigenic response, is a core challenge that is well suited for single cell analysis. Recent years have witnessed transformative and intersecting advances in nanofabrication and genomics that enable deep profiling of individual cells, affording exciting opportunities to study heterogeneity in the immune response at an unprecedented scope. In light of these advances, here we review recent work exploring how immune populations generate and leverage cellular heterogeneity at multiple molecular and phenotypic levels. Additionally, we highlight opportunities for single cell technologies to shed light on the causes and consequences of heterogeneity in the immune system.

Keywords: cellular heterogeneity; immune response; phenotypic variation; single cell genomics.

Figure 1

Schematic of scientific approaches to profile cellular heterogeneity. Conventionally, samples have been subdivided from the ‘top down’ (blue arrows) based marker expression, and cellular subpopulations are iteratively refined. The emerging single cell profiling techniques reviewed here enable a ‘bottom up’ (red arrows) approach to placing cells into functionally distinct groups and to identify novel molecular and phenotypic markers that define them.

Figure 2

Variation in DNA sequence and structure. (a) B and T lymphocytes leverage variability in DNA sequence in order to express myriad antigen receptors from a small set of genetic loci, enabling responses against a wide range of antigens (Figure 2a). (b) Heterogeneity in nucleosome organization and epigenetic state has been shown to drive phenotypic differences in response and survival. While a nascent field, understanding the mechanisms that establish epigenetic heterogeneity and their ramifications for immune cells is an exciting area for future research.

Figure 3

Heterogeneity in gene expression. (a) Traditionally, differences in single cell RNA levels have been probed by RNA-FISH which enables measurement of both RNA expression and its spatial organization. This enables direct assessment of how heterogeneity in one may be couple to that of its neighbors. (b) Higher multiplexing of single cell RNA measurements, for example with single cell qPCR, enables the classification of cells along defined molecular signature. (c) Transcriptome-wide approaches, i.e., single cell RNA-Seq, enable an unbiased approach to find structure in cellular heterogeneity and represent a new systems-biology approach for identifying cell types and reconstructing transcriptional networks.

Figure 4

Cell-to-cell differences in protein levels and activity. (a) Directly assessing the levels and activity of important proteins, e.g., transcription factors, can give insights into timing and magnitude of cellular response. Importantly, fluorescence-based techniques allow for time-lapse measurements of the dynamics of single cell expression and localization (e.g., nuclear translocation). (b) High-throughput, multivariate protein profiling strategies, like FACS, enable exploration and precise quantification of population structure by profiling tens of thousands of cells. (c) The recent development of heavy-metal protein detection strategies (CyTOF, see Box 1) has significantly augmented the number of single cell proteins that can be simultaneously measured. This exciting technology will enable an even more detailed under of the immune cell spectrum, the cellular continuum representing hematopoetic differentiation, and its evolution and in health and disease.

Figure 5

Advantages achieved through immune cell heterogeneity. (a) Cell-to-cell differences engender flexibility and breadth to the immune system, enabling, for example, response to myriad antigens. (b) In spite of tasking different cells with difference portions of the response, the immune system needs to maintain robustness. One way of coordinating the activity of individual cells is through intercellular communication (e.g., with cytokines). This allows individual cellular responses to be communicated and then propagated or restrained. (c) Heterogeneity in cellular expression can also enable complex population level behaviors from simple single cellular decisions. For example, placing different thresholds on cellular activation can translate a digital decision at the single cell level to a graded analog response at the population level.