SummaryThe co-ordination of T-cell motility, adhesion and activation remains poorly understood. It is also unclear how these functions are co-ordinated with external stimuli. Here we unveil a series of molecular interactions in cis at the surface of T lymphocytes with potent effects on motility and adhesion in these cells, and communicating with proliferative responses. These interactions were controlled by the signature cytokines of T helper subsets interleukin-2 (IL-2) and IL-4. Low-density lipoprotein receptor-related protein 1 (LRP1) was found to play a key role for T-cell motility by promoting development of polarized cell shape and cell movement. Endogenous thrombospondin-1 (TSP-1) enhanced cell surface expression of LRP1 through CD47. Cell surface expressed LRP1 induced motility and processing of TSP-1 while inhibiting adhesion to intercellular adhesion molecule 1 and fibronectin. Interleukin-2, but not IL-4, stimulated synthesis of TSP-1 and motility through TSP-1 and LRP1. Stimulation of the T-cell receptor (TCR)/CD3 complex inhibited TSP-1 expression. Inhibitor studies indicated that LRP1 regulated TSP-1 expression and promoted motility through JAK signalling. This LRP1-mediated motogenic signalling was connected to CD47/Gi protein signalling and IL-2-induced signalling through TSP-1. The motogenic TSP-1/LRP1 mechanism antagonized TCR/CD3-induced T-cell proliferation. These results indicate that LRP1 in collaboration with TSP-1 directs a counter-adhesive and counter-proliferative motogenic cascade. T cells seem programmed to prioritize movement before adhesion through this cascade. In conclusion, vital decision-making in T lymphocytes regulating motility, adhesive interactions and proliferation, are integrated through a molecular mechanism connecting different cell surface receptors and their signalling pathways.

SummaryT lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130 000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1-calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T-cell adhesion, and notably also T-cell receptor (TCR) -induced activation, are subject to constant suppression through shedding of low-density lipoprotein receptor-related protein 1 (LRP1). The broad-spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR-induced activation and adhesion to β and β integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full-length thrombospondin-1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non-adhering cells and expression of TSP170 correlated with T-cell motility. The pro-adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP-1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP-1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP-1 in integrin-dependent adhesion and TCR-induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP-1-dependent pathway and ligation of CD28 antagonizes shedding independent of TSP-1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T-cell level.