Dendritic cell chemotaxis in 3D under defined chemokine gradients reveals differential response to ligands CCL21 and CCL19

Abstract

Dendritic cell (DC) homing to the lymphatics and positioning within the lymph node is important for adaptive immunity, and is regulated by gradients of CCL19 and CCL21, ligands for CCR7. Despite the importance of DC chemotaxis, it is not well understood how DCs interpret gradients of these chemokines in a complex 3D microenvironment. Here, we use a microfluidic device that allows rapid establishment of stable gradients in 3D matrices to show that DC chemotaxis in 3D can respond to CCR7 ligand gradients as small as 0.4%, which helps explain how DCs sense lymphatic vessels in an environment where broadcast distance for chemokine diffusion is hindered by convective flows into the vessel. Interestingly, DCs displayed similar sensitivities to both chemokines at small gradients (≤ 60 nM/mm), but migrated more efficiently towards higher gradients of CCL21, which unlike CCL19 binds strongly to matrix proteoglycans and signals without the need for internalization. Furthermore, cells preferentially migrated towards CCL21 when exposed to equal and opposite gradients of CCL21 and CCL19 simultaneously, even when matrix-binding of CCL21 was prevented. Although these ligands have similar binding affinity to CCR7, our results demonstrate that, in a 3D environment, CCL21 is a more potent directional cue for DC migration than CCL19. These findings provide new quantitative insight into DC chemotaxis in a physiological 3D environment and suggest how CCL19 and CCL21 may signal differently to fine-tune DC homing and positioning within the lymphatic system. These results also have broad relevance to other systems of cell chemotaxis, which remain poorly understood in the 3D context.

Fig. 1.

Establishment and characterization of matrix bound and soluble protein gradients in a microfluidic device. (A) Schematic view of one functional unit of the microfluidic device. The cell-matrix mixture was seeded in the center channel (C), while the chemokines/buffers flowed along the side channels (S1 and S2) to create chemical gradients across the center channel (i–iv). Computational results of CCL19 concentration gradients at various times, red and blue correspond to maximum and minimum concentrations, respectively. (i) At t = 0, chemokine solutions were pumped through the side channels (in this case, CCL19 on the right and plain medium on the left). (ii) After 180 min., a steady-state diffusion concentration gradient was reached. (iii) At this time, the cell-matrix mixture was added to the center channel, containing an average chemokine concentration of the two side channels. (iv) After several minutes, the gradient is reestablished within the center channel, before cell tracking began. (B) Temporal gradient establishment of CCL19 from experiment (solid line) and computation (dashed line). (C) Spatial gradient establishment at different time points of CCL19 (top) and CCL21 (bottom) to a collagen-MG matrix using fluorescently tagged chemokines; inserts show confocal images at 300 min. Darkest blue and red lines indicate 0 and 180 min., respectively, with 10-min. intervals shown between. (D) CCL19 (blue) and CCL21 (red) gradients at steady-state from experiment (solid lines) and computation (dashed lines).

Fig. 2.

Dendritic cells display mild chemokinesis to CCR7 ligands. (A) Percentage of migrating cells (%M); *P = 0.0366. (B) Average cell speed; *P < 0.05 and **P < 0.01. (A and B) All values were normalized to controls for each experiment, and comparisons were made using one-way ANOVA and Tukey posttest. (C, D) Histograms of normalized cell speeds in various concentration ranges of (C) CCL19 and (D) CCL21. (A–D) For each data point, n = 2–14, each representing the average of 71–594 cells.

Fig. 3.

Dendritic cell chemosensitivity to CCL21 is greater than to CCL19. (A) Directed migration velocity (V_x) as a function of chemokine gradient (∇C). Solid lines show the fit of data to Eq. 1. (B) V_x as a function of the average concentration (C_Avg). (C) V_x as a function of the relative gradient (∇C/C_Avg). (A–C) For each data point, n = 2 - 14, each representing an average of 70–850 cells. Error bars indicate 95% confidence interval.

Fig. 4.

Dendritic cell migration is preferentially skewed towards CCL21 vs. CCL19. (A) The average velocity V_x of cells in competing gradients of CCL19 and CCL21 as indicated. Dark columns: 1.5 mg/mL collagen ± 10% MG, white columns: collagen only. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to group D with one-way ANOVA and Tukey posttest. Error bars show 95% confidence intervals. For each data point, n = 2, each representing an average of 84–123 cells. (B–E) Computationally estimated concentration gradients corresponding to the scenarios in (A). Lines indicate concentrations of CCL19 (dotted black line), soluble CCL21 (solid gray line), and total CCR7 ligand (solid black line).