Cellular Heterogeneity Based on Microniches

Hematologists and immunologists are often focused on cellular heterogeneity. New markers or transcriptome profiles are ever increasing, breaking down blood and tissue resident cells into smaller and more discrete “cell types.” With the emergence of single-cell RNA sequence profiling, heterogeneity is being discovered in what were thought to be more or less homogenous cell populations.

Recent work from Dr. Alon Oyler-Yaniv and colleagues outlines research strategies for the future, and may help to explain some of this heterogeneity. The premise of their work is that cell-to-cell communication via cytokines in tissues is dependent on the ability of that cytokine to diffuse from the source, and on the number of cells consuming the cytokine. Therefore, in a tissue with a high number of cells that have a receptor for the cytokine (consumers), the relative distribution of a cytokine produced by a cell will be low, creating a small “cytokine niche” (Figure). In contrast, a tissue with a low number of consumers will have more distribution, creating a larger niche.

To test the effect of diffusion-consumption on cytokine responses, the authors chose to use a model of regulatory T cells (Treg) as the consumers, as they have high levels of the high-affinity IL-2 receptor a chain (IL-2Ra). Because normal tissue culture plates have cells at too low of a density to be representative of solid tissue density and to allow the medium to uniformly mix, the authors fabricated their own 96-well plate that they named “the clusterwell plate,” This allowed cell suspensions to be loaded into the plate and then centrifuged to created densely packed cell cultures. Using this clusterwell plate, the authors created cell suspensions with various ratios of Tregs and of CD4 depleted splenocytes, which do not respond to IL-2 and are thus “inert cells” in their model system. This created scenarios where the total number of cells was the same, but the number of consumers was altered. To measure the effects of diffusion-consumption, the authors intracellularly stained for pSTAT5, which is immediately downstream of the IL-2 receptor. The authors demonstrate that as the density of consumers decreased, a larger fraction of the CD4+ IL-2Ra+cells were exposed to IL-2 and were positively stained for pSTAT5. In contrast, as the consumer density increased, pSTAT5 staining decreased. In fact, labeled cells at the bottom of the well were unable to respond to IL-2 when consumer density was higher, demonstrating a restricted cytokine distribution in these cultures.

To visualize these microniches created by diffusion-consumption of IL-2, the authors developed an imaging assay they called PlaneView imaging. The authors mixed either consuming T cells or a combination of 10 percent consumers and 90 percent inert splenocytes with 0.1 percent IL-2–producing T cells. This mixture was then deposited in a monolayer on a glass slide, and 10 more layers of cells without producers were added, creating a three-dimensional system with a small number of producers on the bottom. When pSTAT5 was then imaged, spherical microdomains of IL-2 response were seen. In settings where 100 percent of the cells were consumers, the length scale of the cytokine niche was approximately 3.5 cell diameters, while the 10 percent consumer setting (with the same total number of cells) was approximately 13.5 cell diameters.