Naive T lymphocytes chemotax long distance to CCL21 but not to a source of bioactive S1P.

Naive T lymphocytes traffic through the organism in search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on CCL21 chemokine sensing by CCR7 receptors, while exit into efferent lymphatics relies on sphingolipid S1P sensing by S1PR1 receptors. While both molecules are claimed chemotactic, a quantitative analysis of naive T lymphocyte migration along defined gradients is missing. Here, we used a reductionist approach to study the real-time single-cell response of naive T lymphocytes to CCL21 and serum rich in bioactive S1P. Using microfluidic and micropatterning ad hoc tools, we show that CCL21 triggers stable polarization and long-range chemotaxis of cells, whereas S1P-rich serum triggers a transient polarization only and no significant displacement, potentially representing a brief transmigration step through exit portals. Our in vitro data thus suggest that naive T lymphocyte chemotax long distances to CCL21 but not toward a source of bioactive S1P.

Keratocytes migrate against flow with a roly-poly-like mechanism.

While cell migration can be directed by various mechanical cues such as force, deformation, stiffness, or flow, the associated mechanisms and functions may remain elusive. Single cell migration against flow, repeatedly reported with leukocytes, is arguably considered as active and mediated by integrin mechanotransduction, or passive and determined by a mechanical bias. Here, we reveal a phenotype of flow mechanotaxis with fish epithelial keratocytes that orient upstream or downstream at shear stresses around tens of dyn cm-2. We show that each cell has an intrinsic orientation that results from the mechanical interaction of flow with its morphology. The bulbous trailing edge of a keratocyte generates a hydrodynamical torque under flow that stabilizes an upstream orientation, just as the heavy lower edge of a roly-poly toy generates a gravitational torque that stabilizes an upright position. In turn, the wide and flat leading edge of keratocytes destabilizes upstream orientation, allowing the existence of two distinct phenotypes. To formalize these observations, we propose a simple mechanical model that considers keratocyte morphology as a hemisphere preceded by a wide thin sheet. Our findings show that this model can recapitulate the phase diagram of single cell orientation under flow without adjustable parameters. From a larger perspective, this passive mechanism of keratocytes flow mechanotaxis implies a potential absence of physiological function and evolution-driven process.

Leukocyte transmigration and longitudinal forward-thrusting force in a microfluidic Transwell device.

Transmigration of leukocytes across blood vessels walls is a critical step of the immune response. Transwell assays examine transmigration properties in vitro by counting cells passages through a membrane; however, the difficulty of in situ imaging hampers a clear disentanglement of the roles of adhesion, chemokinesis, and chemotaxis. We used here microfluidic Transwells to image the cells' transition from 2D migration on a surface to 3D migration in a confining microchannel and measure cells longitudinal forward-thrusting force in microchannels. Primary human effector T lymphocytes adhering with integrins LFA-1 (αLß2) had a marked propensity to transmigrate in Transwells without chemotactic cue. Both adhesion and contractility were important to overcome the critical step of nucleus penetration but were remarkably dispensable for 3D migration in smooth microchannels deprived of topographic features. Transmigration in smooth channels was qualitatively consistent with a propulsion by treadmilling of cell envelope and squeezing of cell trailing edge. Stalling conditions of 3D migration were then assessed by imposing pressure drops across microchannels. Without specific adhesion, the cells slid backward with subnanonewton forces, showing that 3D migration under stress is strongly limited by a lack of adhesion and friction with channels. With specific LFA-1 mediated adhesion, stalling occurred at around 3 and 6 nN in 2 × 4 and 4 × 4 µm2 channels, respectively, supporting that stalling of adherent cells was under pressure control rather than force control. The stall pressure of 4 mbar is consistent with the pressure of actin filament polymerization that mediates lamellipod growth. The arrest of adherent cells under stress therefore seems controlled by the compression of the cell leading edge, which perturbs cells front-rear polarization and triggers adhesion failure or polarization reversal. Although stalling assays in microfluidic Transwells do not mimic in vivo transmigration, they provide a powerful tool to scrutinize 2D and 3D migration, barotaxis, and chemotaxis.

Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins.

Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules. Moreover, lymphocytes orient towards increasing adhesion with VLA-4 integrins (also known as integrin α4ß1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLß4), which has not previously been observed. This counterintuitive 'reverse haptotaxis' cannot be explained by existing mechanisms of mesenchymal haptotaxis involving either competitive anchoring of cell edges under tension or differential integrin-activated growth of lamellipodia, because they both favor orientation towards increasing adhesion. The mechanisms and functions of amoeboid adhesive haptotaxis remain unclear; however, multidirectional integrin-mediated haptotaxis might operate around transmigration ports on endothelia, stromal cells in lymph nodes, and inflamed tissue where integrin ligands are spatially modulated.