Background

The mammalian immune system has evolved to identify and limit the spread of potential pathogens while minimizing collateral tissue damage caused by the immune system itself. To achieve this, immune cells rely on a network of cytokine mediators that enable cell-to-cell communications and broadcast information about the magnitude and nature of the pathogenic insult. Vast arrays of different cytokines bind strongly to their cognate receptors, often with characteristic binding affinities in the nano- or pico-molar range. Immunological niches are generated via cytokine communications. For example, in both the bone marrow and the thymus, secretion of Interleukin-7 (IL-7) by stromal cells supports the survival of proliferating B and T cell progenitors, respectively (Tokoyoda et al., 2004; Alves et al., 2009). The size of the cytokine niche controls the number of maturing progenitors, thereby keeping the blood cell compartments in equilibrium (Böyum, 1968; Weist et al., 2015).

We aim to collect information about the spatial and temporal dynamics of cytokines and how these two parameters influence the immune response. This is an area of immunology that is currently under-studied. Many assays test the effects of cytokines in tissue-culture dishes, where media is well-mixed, leading to homogeneous fields of growth and differentiation factors. The intricate and highly specialized architecture of the secondary lymphoid organs sets up niches where cells sense stimuli such as pathogen components and cytokines, proliferate, mature, differentiate, and die. Cytokine concentration gradients are formed within these niches such that some cells have greater or lesser access to cytokines than others (Liu et al., 2015). Measuring how far cytokines spread from their source, and the gradients they form, is key to unravelling the mechanism of the phenotypic heterogeneity of immune cells in differentiation, proliferation, and death (Feinerman et al., 2010; Busse et al., 2010; Höfer et al., 2012; Müller et al., 2012; Thurley et al., 2015).

Due to the typically low concentrations (pM range) of free cytokines in vivo, direct measurement of cytokine fields is difficult at best and maybe impossible. However, due to their high sensitivity to cytokine and graded, concentration-dependent response, the signaling levels of cells in response to cytokines can itself be used as a bio-sensor for cytokine concentrations (Oyler-Yaniv et al., 2017).

In this protocol, we describe how to directly image the signaling response generated around a cytokine producer in vitro, in conditions that mimic in vivo conditions: high cell density and no convection. Our method is general and can be applied to any cell type and any diffusible stimulus, and only depends on the existence of a specific antibody to target the downstream signaling molecule of interest and/or of live cell reporters.