Dendritic cell (DC) migration to draining lymph nodes (dLNs) is crucial for initiating adaptive immunity and maintaining tolerance. This migration depends on the chemokine CCL21, which exists in two proteoforms: a full-length, tissue-immobilized variant and a cleaved, soluble variant (CCL21-ΔC). Despite its crucial role in DC migration, the mechanisms behind CCL21 cleavage and the impact of the cleaved proteoform on DC migration remain unclear. This study aims to elucidate whether plasmin, generated from its precursor plasminogen on the surface of lymyphatic endothelial cells (LECs) in a urokinase plasminogen activator (uPA)-dependent manner, regulates CCL21 cleavage and affects DC migration.

We investigated the regulation of CCL21 cleavage in the skin by the plasmin/uPA/uPAR system. We employed genetic deletion of uPA and its binding to its receptor uPAr, alongside analyzing perilymphatic CCL21 gradients and DC migration by whole-mount immunofluorescence in both steady-state and inflamed conditions. CCL21 cleavage products were examined using WB and ELISA, while DC migration was assessed using in vivo and ex vivo migration studies. The presence of CCL21 and CCL21-ΔC in the dLNs was investigated using immunofluorescence staining of LN sections and protein analysis tecniques.

Our findings reveal that the immobilized perilymphatic CCL21 gradient in the skin is continuously remodeled by plasmin which is generated on LECs via a uPA-dependent pathway. Deletion of uPA or its binding to uPAr leads to CCL21 accumulation around LVs and reduced DC migration into LVs. CCL21 cleavage occurs under steady-state conditions but is enhanced during inflammation, resulting in generation of more CCL21-ΔC with higher chemotactic activity. In contrast, CCL21 in dLNs is mostly full-length and less susceptible to uPA/uPAR/plasmin-mediated cleavage, ensuring stable DC migration.

We found that LECs in the skin, but not in dLNs, are the sites of constant CCL21 production and uPA/plasmin-mediated CCL21 cleavage. Enhanced CCL21 cleavage during inflammation increases the chemotactic activity of CCL21-ΔC, facilitating a robust immune response. The tissue-specific control of CCL21 gradients by this system optimizes DC migration to dLNs. These findings provide new insights into the molecular mechanisms governing DC migration and may inform new therapeutic strategies targeting immune responses