A Tunable Diffusion-Consumption Mechanism of Cytokine Propagation Enables Plasticity in Cell-to-Cell Communication in the Immune System

Abstract

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80-120 μm, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non-genetic cell-to-cell variability that is often overlooked in single-cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors.

Keywords: Cell-to-cell communications; Cell-to-cell variability; Cytokine niches; Cytokines; Diffusion/Consumption; Interleukin-2; Quantitative Immunology; STAT5; Theoretical Modeling.

Figure 1. The Treg cell compartment is dynamic during an immune challenge

Recipient mice were adoptively transferred with TCR transgenic T cells and immunized. Animals were euthanized after 2 days and the size of the Treg cell compartment was measured. Bars show the relative size of the Treg cell compartment in naïve and in immune activated mice, in the lymph nodes and in the spleen. Results are from experiment using 5 naïve mice and 4 immune activated mice. Data shown as (mean ± SEM).

Figure 2. Diagram of simple diffusion-consumption kinetics

Cytokines are secreted by a producing cell, and freely diffuse between cells. Upon binding to a receptor the cytokine is consumed. This creates a gradient of localized cytokine niche with a typical length scale λ_niche. Increasing consumption, either by increasing the density of consuming cells, n_consumers, or by enhancing the single-cell consumption rate, k_consumption, will lead to a decrease in λ_niche and vice versa. When λ_niche is small compared to the size of the system, we expect an increase in cell-to-cell variability.

Figure 3. Cytokine penetration in dense conditions is explained by a diffusion-consumption mechanism

(A) The pSTAT5 response of CD4⁺IL-2Rα₊ consuming cells to different doses of IL-2 in well mixed conditions. The dashed line represents a threshold set at 20% of the maximum MFI response. Cells with levels of pSTAT5 higher than the threshold are considered exposed to cytokine and counted at pSTAT5⁺. (B) Diagram of experimental design. (C) Distributions of pSTAT5 in IL-2Rα₊ cells in clusterwells for different ratios of consumers to inert cells. (D) The percent pSTAT5⁺ cells of IL-2 consumers in the clusters, and the theoretically predicted scaling. Data shown as (mean ± SEM), results from an experiment using three experimental replicates. The data are representative of at least three additional independent experiments. See also Figs. S2, S3.

Figure 4. Micro domains of signaling cells are generated around cytokine sources

(A) Immunofluorescence staining of cell preparations containing either 100% IL-2Rα⁺ consuming cells or 10% consuming cells and 90% IL-2Rα⁻ inert cells, and a small number (<0.01%) of IL-2 producing T cells in a PlaneView imaging device. (See Fig. S4 for more examples). (B) A profile of the pSTAT5 response was generated as a function of distance from the central producer by segmenting individual cells and measuring pSTAT5 on a single cell level. The profiles were then fit with the theoretical solution of the 3D diffusion-consumption equation. The signaling length-scale λ_niche was extracted as a fitting parameter. The data shown is aggregated from 34 micro domains for the 100% condition and 17 for the 10%. Data shown as (mean ± SEM). (C) Fitted signaling length-scales λ_niche are 3.5 ± 0.2 cell diameters for 100% consumers and 13.5 ± 1.6 for 10% consumers. Black dashed line are the predicted values based on average parameters, grey dashed lines are extreme values given the biological range of parameters. (D) The average concentration of IL-2 at a given distance from a producing cell was inferred from the pSTAT5 profiles for different densities of consumers. See section 3.2.1 of the supplemental materials for full details of the procedure. See also Figs. S4.

Figure 5. Boosting the Treg cell compartment results in decreased fractions of IL-2 responding cells after immunization

(A) Diagram of experimental design - B10.A recipient mice were boosted as described in the methods. Boosted and wild type mice were adoptively transferred with either 5×10⁵ or 5×10⁶ 5C.C7 CD4⁺ T cells. Transferred mice were immunized, and then rapidly euthanized 6 hours later. Spleens and lymph nodes were reserved for either flow cytometric pSTAT5 analysis or I.F. microscopy. (B) Representative histograms of pSTAT5 among Treg cells from flow cytometry. pSTAT5⁺ cells were gated based on the dashed threshold line drawn. (C) The fraction of pSTAT5⁺ Treg cells is lower for IL-2i.c. boosted mice. Bar plots of pSTAT5⁺ Treg cells are from an experiment using three mice per group. The data are representative of three independent experiments. See also Figs. S5, S6.

Figure 6. The size and shape of signaling micro domains in vivo, are explained by a tunable diffusion-consumption mechanism

Immunofluorescence staining of spleen nodule (A) and lymph nodes (B) sections from wild type (middle) and IL-2 i.c. boosted (right) mice after peptide immunization, and of lymph node section from Foxp3-eGFP mouse at homeostasis (left). (C) pSTAT5 autocorrelation functions were calculated for the immunized IL-2 i.c. boosted and wild type B10.A mice, and for Foxp3-eGFP B6 mice at homeostasis, and fit to a decaying exponential (See section 3.3 of Sup. Mat. for details). The correlations decay coefficient is the signaling length scale λ_niche. Results quantify an experiment using three mice per group and 3–4 lymph nodes per mouse. Whenever errors do not appear they are smaller than markers. (D) Measured signaling length scales for the three different mouse groups, and for in vitro samples (Fig. 4), and estimations based on known microscopic parameters. Black dashed line are the predicted values based on average parameters, grey dashed lines are extreme values given the biological range of parameters. See also Figs. S5, S6.