Neuroserpin: a regulatory role for a neuronal serpin in human T cell activation and migration

Abstract

Neuroserpin is an axonally secreted serine protease inhibitor, or serpin, which inhibits trypsin-like serine proteases. Neuroserpin most effectively inhibits tissue plasminogen activator. Most research has focussed on the function of neuroserpin in the nervous system. Recently, it was discovered that neuroserpin is also expressed by T cells and immune cells of the myeloid lineage. The overall goal of this thesis is to identify the function of neuroserpin in T cell activation and migration. The data presented in this thesis shows, for the first time, that regulation of the proteolytic environment by neuroserpin impacts T cell activation and processing of the homing chemokine CCL21. qPCR, western blotting and immunocytochemistry showed that neuroserpin is expressed in human T cells and partially co-localizes with a TGN38/LFA-1-positive vesicle population and relocates to the synapse upon T cell activation. TGN38 has been proposed as a cargo receptor involved in the secretion of proteins involved in cell adhesion or migration. siRNA-mediated neuroserpin mRNA knockdown during T cell activation increased T cell-T cell adhesion, T cell proliferation, IL-10 secretion and shedding of the Fas ligand. The increased T cell-T cell adhesion was blocked by addition of recombinant neuroserpin or the plasmin-specific inhibitor α2-antiplasmin, demonstrating that the increased interaction is a plasmin-mediated process. Inhibition of ROCKII, a signalling molecule linked to immunological synapse stability through myosin light chain phosphorylation, likewise prevented increased T cell-T cell adhesion. Knockdown of neuroserpin mRNA also increased cleavage of the plasmin/plasminogen/tPA receptor annexin A2 in resting T cells, possibly priming T cells for increased T cell-T cell interaction during activation through increased formation of F-actin. Neuroserpin also regulated the migratory capacity of human T cells and dendritic cells by inhibiting the cleavage of the chemokine CCL21. Cleavage of the C-terminal anchoring peptide in CCL21 enables its chemotactic activity. Neuroserpin prevented T cell- and dendritic cell-mediated processing of the anchoring peptide through inhibition of tissue plasminogen activator. Tissue plasminogen activator did not cleave CCL21 directly but enhanced CCL21 C-terminal processing through generation of plasmin from plasminogen, which was confirmed by LC-MS/MS. The data presented here illustrates the importance of the proteolytic environment in T cell biology.