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.D endritic cells (DCs) are considered the most potent and professional antigen-presenting cells. DCs are positioned throughout the periphery, and when activated, migrate to lymphatic vessels and into lymph nodes, where they can direct antigen-specific Tcell responses. Upon activation or maturation, DCs upregulate the C-C chemokine-receptor CCR7, which allows them to sense and home towards CCR7 ligand-secreting lymphatic vessels and lymph nodes (1). The two known ligands for CCR7 are the C-C chemokines CCL21 and CCL19 (2); both are secreted by stromal cells in the lymph node paracortex to properly position DCs with CCR7 þ naïve T cells for their activation. CCL21, but not CCL19, is also expressed by the endothelium of lymphatic vessels in the periphery (1). Thus, CCR7-mediated chemotaxis is critical for DC homing to, and positioning within, lymph nodes and for T cell activation there (3).CCL19 and CCL21 function as directional signals presumably by virtue of concentration gradients (∇C) that guide DCs towards areas of increasing concentrations. Interestingly, CCL19 and CCL21 have similar binding affinities for CCR7 (4-6) and similar chemotactic potential for DCs and T cells under 2D conditions (7,8), but differ in their internalization (8, 9) as well as their binding affinity to extracellular matrix (ECM) proteoglycans. Specifically, the positively charged C t...

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