Live-Cell Microscopy Reveals That Human T Cells Primarily Respond Chemokinetically Within a CCL19 Gradient That Induces Chemotaxis in Dendritic Cells

Abstract

The ability to study migratory behavior of immune cells is crucial to understanding the dynamic control of the immune system. Migration induced by chemokines is often assumed to be directional (chemotaxis), yet commonly used end-point migration assays are confounded by detecting increased cell migration that lacks directionality (chemokinesis). To distinguish between chemotaxis and chemokinesis we used the classic "under-agarose assay" in combination with video-microscopy to monitor migration of CCR7+ human monocyte-derived dendritic cells and T cells in response to a concentration gradient of CCL19. Formation of the gradients was visualized with a fluorescent marker and lasted several hours. Monocyte-derived dendritic cells migrated chemotactically towards the CCL19 gradient. In contrast, T cells exhibited a biased random walk that was largely driven by increased exploratory chemokinesis towards CCL19. This dominance of chemokinesis over chemotaxis in T cells is consistent with CCR7 ligation optimizing T cell scanning of antigen-presenting cells in lymphoid tissues.

Keywords: CCR7; T cell and mDC co-culture; cell migration; chemokines; gradient formation; real-time microscopy.

Figure 1

Dendritic cells respond chemotactically to a cytokine gradient in the "under-agar" assay, which lasts for several hours as shown by fluorescent dextrans. (A) Images showing the diffusion of fluorescent dextran (blue) a nd migration of mDCs (red) at one, three and five hours of incubation. (B) The formation of a gradient in the under-agar assay as shown by diffusion of 10 kDa dextrans. (C) Graph showing the number of tracked mDCs over time in response to a CCL19 gradient or no chemokine (control). (D) Graph showing the track straightness of mDCs over time in response to a CCL19 gradient or no chemokine (control). (E) Graph showing the displacement of mDCs in the direction of the CCL19 gradient or in the same direction without chemokine (control). (F) "Spider plots" showing the tracks of mDCs over time in response to a CCL19 gradient or no chemokine (control) plotted from a single origin point (red dot). All data is from one representative experiment that was repeated at least three times.

Figure 2

Static assays do not clearly distinguish between chemotaxis and chemokinesis. Images showing migration after 16 hours of incubation of mDCs (red) and T cells (green) without chemokine (Control), in response to a CCL19 gradient, and 100 ng mL^-1 uniform CCL19 concentration.

Figure 3

Unlike DCs, T cells do not show a strong chemotactic response to a CCL19 gradient. "Spider plots" showing the tracks of mDCs and T cells over time in response to a CCL19 gradient or a uniform 100 ng mL^-1 CCL 19 concentration plotted from a single origin point (red dot). All data is from one representative experiment that was repeated at least three times.

Figure 4

Migration behavior of DCs is not altered by the presence of T cells while the number of T cells and their displacement toward CCL19 is increased in the presence of mDCs. (A) Graphs showing the number of tracked cells, track straightness and track speed of mDCs and T cells over time in response to a CCLl9 gradient or uniform 100 ng mL^-1 CCL19 concentration. (B) Graphs showing the displacement of mDCs and T cells towards a gradient of CCL19. The displacement was analyzed by taking the average displacement of T cells toward a CCL19 gradient and subtracting the average displacement of T cells in the same direction in a uniform CCL19 concentration. Data are combined from three independent experiments and are presented as mean +/­ SEM. Results were analyzed using multiple t-tests with the assumption that all populations have the same scatter and was corrected for multiple comparisons using the Holm-Sidak method. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001.